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INTRODUCTION |
Intermediate conductance, Ca2+-activated
K+ channels play crucial roles in a wide array of
physiological processes, including agonist-mediated transepithelial
Cl
secretion across airway and intestinal epithelia
(1-6). Indeed, Dharmsathaphorn and Pandol (7) initially proposed that
Ca2+-mediated intestinal Cl
secretion was
dependent upon the activation of a basolateral membrane K+
conductance in the absence of a change in apical membrane
Cl
conductance. As a consequence of the increased
K+ conductance there would be a hyperpolarization of the
membrane potential, thereby increasing the electrochemical driving
force for Cl
exit across the apical membrane via
constitutively active Cl
channels (7, 8). An increase in
intracellular Ca2+ alone can stimulate Cl
secretion across the colonic epithelial cell line, T84 (5, 9). Yet,
there is a disparity between the magnitude and time course of the rise
in intracellular Ca2+ and the subsequent Cl
secretory response (1, 4) such that the Cl
secretory
response is transient in nature. These data suggest that second
messengers other than Ca2+ may down-regulate the secretory
response. Various inhibitory second messengers have been proposed,
including protein kinase C (5, 10, 11), inositol
tetrakisphosphate (12, 13), and arachidonic acid
(AA)1 (2, 14, 15). It is
proposed that these second messengers may inhibit the K+
conductance, thus modulating the Cl
secretory response
even in the presence of elevated intracellular Ca2+ levels.
Ca2+-mediated agonists are known to increase AA levels in a
wide range of tissues where hIK1 is expressed, including colon and lung
(14, 16-18). This can occur in several ways (19), including: 1)
Ca2+ directly activating PLA2, 2) either
diacylglycerol itself or protein kinase C activating
PLA2, or 3) diacylglycerol lipase directly generating AA
from diacylglycerol. Thus, the generation of AA by
Ca2+-mediated agonists would be expected to lag behind the
rise in intracellular Ca2+. Given this, an effect of AA on
hIK1 would be temporally appropriate to explain the dissociation
between changes in intracellular Ca2+ and the resultant
Cl
secretory response. Likewise, AA is released during
inflammatory responses such as asthma (20) and irritable bowel disease
(21) such that AA may play an important role in modulating ion channels in these diseases.
Arachidonic acid has been shown to modulate a wide variety of ion
channels, including K+, Na+, Ca2+,
and Cl
channels (22-25). Indeed, we previously
demonstrated that inhibition of cytosolic PLA2 resulted in
a potentiated Cl
secretory response to the
Ca2+-mediated agonist, carbachol in T84 cells and that AA
was a potent negative modulator of the intermediate conductance,
Ca2+-dependent K+ channel in these
cells (2). Recently, we (26) and others (3) have confirmed the
molecular identity of this colonic epithelial K+
channel as being the recently cloned hIK1/hSK4 (27, 28). hIK1 is
a member of the KCNN gene family, exhibiting
significant homology with the SK channels (SK1-3), having ~40%
identity at the amino acid level (27, 28).
In the present study, we demonstrate that AA directly inhibits
heterologously expressed hIK1, whereas rSK2 is insensitive to AA. Using
a series of hIK1/rSK2 chimeras and point mutations we demonstrate that
AA inhibits hIK1 via an interaction with two pore-lining amino acids,
Thr250 and Val275. Importantly, substitution of
these amino acids into rSK2 induces sensitivity to AA, confirming the
critical nature of these amino acids in defining the molecular binding
site for AA on hIK1.
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MATERIALS AND METHODS |
Cell Culture--
Human embryonic kidney (HEK293) cells were
obtained from the American Type Culture Collection (Manassas, VA) and
cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad,
CA) supplemented with 10% fetal bovine serum and 1%
penicillin-streptomycin in a humidified 5% CO2/95%
O2 incubator at 37 °C. Cells were transfected using
LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. Stable cell lines were generated for all constructs by
subjecting cells to antibiotic selection (1 mg/ml G418) 48 h
post-transfection. Selection was typically complete within 14 days
post-transfection. Following selection, the concentration of G418 was
reduced to 0.2 mg/ml. We and others have previously demonstrated that
hIK1, heterologously expressed in HEK cells, has identical biophysical,
pharmacological, and second messenger-dependent regulatory
properties to the endogenously expressed channel (26, 29, 30)
demonstrating the utility of this heterologous expression system.
Molecular Biology, Generation of Chimeras, and Site-directed
Mutagenesis--
pBF plasmid containing the cDNAs for full-length
hIK1 and rSK2 were kindly provided by J. P. Adelman (Vollum Institute,
Oregon Health Sciences University). hIK1 and rSK2 were subcloned into pcDNA3.1+ (Invitrogen) using the EcoRI and
XhoI restriction sites. The schematic structures of hIK1 and
rSK2 are shown in Figs. 1 and 2, respectively.
Chimera generation and site-directed mutagenesis were performed as
described by Gerlach et al. (31). Briefly, chimeras between hIK1 (427 amino acids) and rSK2 (580 amino acids) were generated by
overlap extension polymerase chain reaction using Pfx
polymerase (Invitrogen). The chimeras 26IK-SK, 200IK-SK, SK-200IK, and
SK-287IK were generated (see Fig. 3 for schematic representations of
these chimeric constructs). The chimeras were subcloned into the
pcDNA 3.1+ vector using EcoR1 and
XhoI restriction sites. Point mutations were generated using
the QuikChange site-directed mutagenesis strategy using Pfu
polymerase (Invitrogen). The fidelity of all constructs utilized in
this study were confirmed by sequencing (ABI PRISM 377 automated
sequencer, University of Pittsburgh) and subsequent sequence alignment
(NCBI BLAST 2.0) with hIK1 (GenBankTM accession
number AF022150) and rSK2 (GenBankTM accession number
U69882). Stable cell lines of each of these constructs were established
in HEK293 cells as described above.
Electrophysiology-Inside/Out Patch-Clamp Experiments--
The
effects of AA on hIK1, rSK2, chimeric, and point-mutation channels were
assessed with excised, inside-out patch-clamp experiments as a
functional assay. Currents were recorded using a List EPC-7 amplifier
(Medical Systems, Greenvale, NY) and stored on videotape for later
analysis. Electrodes were fabricated from thin-walled borosilicate
glass (World Precision Instruments, Sarasota, FL), pulled on a vertical
puller (Narishige, Long Island, NY), fire-polished, and had a
resistance of 1-4 M
.
During patch-clamp experiments, the bath solution was (in
mM) potassium gluconate 145, KCl 5, MgCl2 2, HEPES 10, CaCl2 10 µM, pH 7.2 (adjusted with
KOH). To obtain a 0 Ca2+ bath solution EGTA (1 mM) was added without CaCl2 (estimated free
Ca2+ <10 nM). All bath solutions contained 100 µM 1-ethyl-2-benzimidazolinone to maximally activate
channels and 300 µM ATP to prevent channel rundown (31).
The pipette solution was (in mM) potassium gluconate 140, KCl 5, MgCl2 1, HEPES 10, CaCl2 1, pH 7.2 (adjusted with KOH). All chimeras tested were strictly
Ca2+-sensitive as a Ca2+-free bath solution
eliminated all channel activity. Generally, a 3-µM
concentration of AA was used (except for the concentration-response experiments). Constructs that were not sensitive to block by AA were
always tested in parallel with hIK1 or a chimera known to be sensitive
to AA as a positive control. All experiments were performed at room
temperature. All patches were held at a holding potential of
100 mV.
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 the baseline in all recordings. Channel data were digitized with
the Fetchex application within the pCLAMP suite of programs (version
6.04, Axon Instruments, Foster City, CA) using a PC computer.
Single channel analysis was performed on records after low pass
filtering at 200 Hz and sampling at 500 Hz (Digidata 1200, Axon
Instruments). Total channel current was determined using Biopatch
software (version 3.3, Bio-Logic).
Chemicals--
1-Ethyl-2-benzimidazolinone, AA,
5,8,11,14-eicosatetraynoic (ETYA), clotrimazole, and all general
chemicals were obtained from Sigma, unless otherwise stated. ATP was
purchased from Roche Applied Science and added directly to a Ringer's
solution as a dry powder. 1-Ethyl-2-benzimidazolinone, AA, and
clotrimazole were made as 10,000-fold stock solutions in
Me2SO.
Statistics--
All data are presented as means ± S.E.,
where n indicates the number of experiments. Statistical
analysis was performed using a Student's t test (paired or
unpaired) or analysis of variance with Student-Newman-Keuls multiple
posttest. A value of p < 0.05 is considered
statistically significant and is reported.
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RESULTS |
Arachidonic Acid Inhibits Heterologously Expressed
hIK1--
We previously demonstrated that AA inhibits endogenously
expressed hIK1 in T84 cells with high affinity (2). Therefore, we
initially characterized the AA sensitivity of heterologously expressed
hIK1 (Fig. 1A) in HEK293 cells
in excised, inside-out patches. As shown in a representative experiment
in Fig. 1B, following the establishment of a stable current,
the addition of AA (3 µM) induced a significant reduction
in current. The average current prior to AA was 468 ± 60 pA,
which was reduced by 82 ± 2% to 81 ± 18 pA in the presence
of AA (p < 0.001, n = 16). Arachidonic acid inhibited hIK1 in a concentration-dependent manner,
being half-maximal (IC50) at 1.4 ± 0.7 µM (n = 5, Fig. 1C), a value similar to what we previously reported for endogenously expressed hIK1
(IC50 = 0.42 µM) in T84 cells (2).

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Fig. 1.
Effect of arachidonic acid and ETYA on
hIK1. A, schematic of hIK1. B,
representative current (pA) trace of the effect of arachidonic acid (3 µM) on hIK1. hIK1 was heterologously expressed in HEK
cells and recorded in excised, inside-out patches at a holding
potential of 100 mV. C, concentration-response inhibition
curve of arachidonic acid on hIK1 (n = 5). Data are
plotted as the percent current remaining in the presence of a given
concentration of arachidonic acid. Data were fitted with the
Michaelis-Menten equation being half-maximally inhibited at 1.4 µM. D, effect of ETYA (10 µM)
and AA (3 µM) on the channel current of hIK1.
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Direct Effect of Arachidonic Acid on hIK1--
Arachidonic acid
can elicit its effect by interacting directly with ion channels or
indirectly by metabolites produced via cyclo-oxygenase (COX),
lipoxygenase (LOX), and/or cytochrome P450 pathways (22, 23). We
previously demonstrated that inhibitors of COX, LOX, and cytochrome
P-450 did not affect the AA-dependent inhibition of
endogenously expressed hIK1 in T84 cells (2). We confirmed these
results in the present study by examining the effects of AA in the
presence of ETYA (10 µM), a blocker of COX, LOX, and P450
pathways (32, 33) using excised, inside-out patches. As shown in Fig.
1D, ETYA had no effect on hIK1 current. Under control
conditions channel current was 622 ± 83 pA, and ETYA did not
significantly reduce this current (560 ± 94 pA). However,
subsequent addition of AA (3 µM) reduced channel current by 77% (117 ± 26 pA, p < 0.01, n = 3) in the continued presence of ETYA. These data
suggest that oxidative metabolites of AA are not involved in the
inhibition of hIK1 observed.
rSK2 Is Insensitive to Inhibition by Arachidonic Acid--
Since
hIK1 shares ~40% sequence homology with rSK2 (27, 28) we examined
whether rSK2 could be inhibited by AA in excised, inside-out patches. A
representative experiment is shown in Fig. 2B in which AA did not reduce
channel current in HEK cells heterologously expressing rSK2. The
average channel current was 491 ± 169 pA in the absence of AA and
438 ± 136 pA in the presence of AA (n = 4),
representing an 8 ± 3% inhibition of current flow. The
Ca2+ dependence of rSK2 was subsequently verified by
demonstrating that our 0 Ca2+ solution reduced channel
current to zero (Fig. 2B). These data demonstrate that even
though hIK1 and rSK2 share ~40% sequence homology AA sensitivity is
specific to hIK1.

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Fig. 2.
Effect of arachidonic acid on rSK2.
A, schematic of rSK2. B, representative current
(pA) trace of the effect of arachidonic acid (3 µM) on
rSK2. rSK2 was heterologously expressed in HEK cells and recorded in
excised, inside-out patches at a holding potential of 100 mV. 0 Ca2+ steps occurred at the beginning and the end of the
experiment to illustrate the Ca2+ dependence of rSK2.
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Arachidonic Acid Sensitivity of hIK1 Is Localized to the S5-Pore-S6
Region--
Based on our observation that AA inhibits hIK1, while
having no effect on rSK2, we used a chimeric hIK1/rSK2 strategy to
identify the region of the channel that confers AA sensitivity to hIK1. Wang and co-workers (34) have demonstrated that the
NH2 terminus (particularly Ser4) of
ROMK1 plays a key role in the determination of the AA effect on that
channel. hIK1 has a serine (Ser24) just prior to S1. Thus,
we initially generated the chimera 26IK-SK (Met1-Ala26 of hIK1 with
Asp137-Ser508 of rSK2; schematic diagram in
Fig. 3A) in which only the
cytoplasmic NH2 terminus is derived from hIK1. As shown for
one experiment in Fig. 3A (right panel), after a
sustained current was established, AA had little effect (7 ± 3%
inhibition, Fig. 3E) on this construct; however, 0 bath
Ca2+ eliminated channel current. In five experiments, the
channel current prior to the addition of AA was 296 ± 16 pA and
277 ± 17 pA in the presence of AA. The absence of inhibition of
the 26IK-SK channel suggests that the cytoplasmic NH2
terminus of hIK1 is insufficient to confer AA sensitivity to
rSK2.

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Fig. 3.
S5-pore-S6 region of hIK1 confers arachidonic
acid sensitivity. Schematics of the 26IK-SK (A),
SK-287IK (B), 200IK-SK (C), and SK-200IK
(D) chimeras in which open circles represent
amino acids from hIK1 and solid circles represent amino
acids from rSK2. Single amino acid nomenclature and arrows
indicate the hIK1 amino acid that defines the chimeric junction. Also
shown are representative current (pA) traces of chimeras 26IK-SK
(A), SK-287IK (B), 200IK-SK (C), and
SK-200IK (D) in response to 3 µM AA.
E, percentage inhibition summary data for all experiments
for the chimeric channels are given (number of experiments shown above
the bars). Data for hIK1 and rSK2 are also presented for
comparison. Chimeras were heterologously expressed in HEK cells and
recorded in excised inside-out patches at a holding potential of 100
mV. 0 Ca2+ steps occurred at the beginning and the end of
the experiment to illustrate the Ca2+ dependence of the
chimeric channels.
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Kim et al. (35) reported that the COOH terminus is important
in the AA sensitivity of TREK-2, a two-pore, four-transmembrane domain
K+ channel. To elucidate the role of the COOH terminus in
the AA sensitivity of hIK1, we generated the chimeric channel SK-287IK (Met1-Ala395 of rSK2 with
Arg287-Lys427 of hIK1; see Fig. 3B
for schematic) in which only the cytoplasmic COOH terminus is derived
from hIK1. As shown in Fig. 3B (right panel), AA
did not alter the channel current of the SK-287IK channel. In six
excised, inside-out patch-clamp experiments, the average current was
467 ± 169 pA in the absence of AA and 472 ± 176 pA in the
presence of AA. The failure of AA to reduce the activity of the
SK-287IK channel suggests that the cytoplasmic COOH terminus of hIK1 is
not sufficient to confer AA sensitivity to rSK2.
To determine whether the S1-S4 or pore region of hIK1 was important in
the AA sensitivity, we generated two additional channel constructs that
alternated the first half of hIK1 and rSK2. The first construct,
200IK-SK (Met1-Met200 of hIK1 with
Thr207-Ser508 of rSK2; see Fig. 3C
for schematic) is composed of the entire NH2 terminus to
the beginning of S5 of hIK1, while the remainder of the channel
construct is derived from rSK2. As shown for one representative
experiment in Fig. 3C (right panel), AA only
modestly reduced current flow through 200IK-SK. AA inhibited the
channel current of 200IK-SK by an average of only 5 ± 5%, from
239 ± 33 pA to 228 ± 37 pA (n = 8, Fig.
3E). The second channel construct was SK-200IK
(Met1-Leu306 of rSK2 with
Met200-Lys427 of hIK1; see Fig. 3D
for schematic) in which the NH2 terminus through the
beginning of S5 is from rSK2, whereas S5-pore-S6 and the COOH terminus
is derived from hIK1. As shown by one representative trace in Fig.
3D (right panel), AA significantly reduced the
channel current of SK-200IK. In four experiments the average current of SK-200IK was reduced an average of 70 ± 5% (p < 0.05, Fig. 3E) from 430 ± 109 pA in the absence of AA
to 127 ± 34 pA in the presence of AA. In light of the data for
the SK-287IK channel construct (Fig. 3B), which demonstrated
that the COOH terminus was not crucial for the AA inhibition of hIK1,
the experimental results of the SK-200IK channel construct confirm that
the AA sensitivity of hIK1 lies within the S5-pore-S6 region of hIK1.
Multiple Amino Acid Mutations of the S5 Linker-Pore Region of
hIK1--
To identify the specific amino acid residue(s) responsible
for the inhibition of hIK1 by AA, we made selected amino acid mutations in hIK1 with their rSK2 amino acid counterparts. The amino acid alignment of the S5 linker-pore region is shown in Fig.
4. The amino acids that are different
between hIK1 and rSK2 are shown in bold type. Initially,
three multiple amino acid mutations of hIK1 were generated. These
mutations are shown in Fig. 4 (underlined) and include: GHL
(G235S/H236N/L237F), SDTL (S238L/D239G/T240A/L241M), and VGMW
(V256M/G259N/M261Y/W262C). Fig. 5
illustrates representative current traces for GHL/SNF (5A), SDTL/LGAM
(5B) and VGMW/MNYC (5C). For each of these channels, AA significantly
(p < 0.05) inhibited current flow (GHL/SNF, 623 ± 199pA to 133 ± 49 pA, n = 5, Fig,
5A; SDTL/LGAM 494 ± 115 pA to 83 ± 19 pA,
n = 6, Fig. 5B; VGMW/MNYC 246 ± 85 pA
to 53 ± 19 pA, n = 5, Fig. 5C). These data are summarized in the bar graph shown in Fig. 5F and
suggest that these amino acids are not important in conferring the AA sensitivity of hIK1.

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Fig. 4.
Amino acid sequence of hIK1 and rSK2 for the
S5 linker-pore region. The amino acid sequences for hIK1
(Gln229-Cys267) and rSK2
(Asp338-Cys376) are provided in single-letter
code. The selectivity filter (GYGD) is indicated by a dashed
box while the pore-helix is indicated by an open box.
The S5 and S6 transmembrane domains are prior to Gln229 and
subsequent to Cys267, respectively. Amino acid residues
that are not similar for hIK1 and rSK2 are in bold type. The
underlined residues comprise the mutated hIK1 constructs in
which the appropriate aligned residue of rSK2 was substituted into
hIK1. These mutations include: GHL/SNF, SDTL/LGAM, VGMW/MNYC, and
T250S.
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Fig. 5.
Effect of AA on the channel current of
mutated hIK1-Thr250 and -Val275 confer AA
sensitivity. Representative current (pA) traces of the mutated
hIK1 channels with GHL/SNF (A), SDTL/LGAM (B),
VGMW/MNYC (C), T250S (D), and V275A
(E) in response to 3 µM AA. F,
percentage inhibition summary data for all experiments for the mutated
channels are given (number of experiments shown above the
bars). Data for hIK1 are also presented for comparison.
Mutated hIK1 channels were heterologously expressed in HEK cells and
recorded in excised, inside-out patches at a holding potential of 100
mV. 0 Ca2+ steps occurred at the end of the experiments for
the T250S and V275A constructs to illustrate the Ca2+
dependence of these channels.
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Mutations of Thr250 and Val275 Selectively
Abolish AA Sensitivity of hIK1--
We next mutated the threonine
(Thr250) just prior to the K+ selectivity
filter GYG (Gly-Tyr-Gly) motif of hIK1 to serine (T250S-hIK1), which is
present in rSK2 (see Fig. 4). A representative excised patch-clamp
experiment of this construct is shown in Fig. 5D. The
T250S-hIK1 channel possessed significantly reduced (p < 0.05) AA sensitivity compared with hIK1 (Fig. 1B).
Arachidonic acid reduced the channel current of this construct by only
8 ± 2% from 304 ± 59 pA to 280 ± 62 pA (Fig.
5F, n = 8). The effect of AA on the
T250S-hIK1 channel was not different than that for rSK2 (8 ± 3%
inhibition, Fig. 2). These data suggest that Thr250 is the
critical amino acid responsible for conferring the AA sensitivity to hIK1.
It is interesting to note that Chandy and co-workers (36) previously
reported that Thr250, in combination with
Val275 (within S6), of hIK1 are crucial in conferring the
clotrimazole sensitivity of hIK1. Based on the crystal structure of the
KCSA K+ channel (37), Chandy and co-workers (36) postulated
that Thr250 and Val275 line the water-filled
pocket that lies just below the narrow K+ selectivity
filter of hIK1. In light of the data from our T250S-hIK1 construct, it
was of interest to determine whether Val275 played a
similar role in the AA sensitivity of hIK1. Therefore, we generated
separate hIK1 constructs with V275A or the double mutation T250S/V275A
to assess the effect of these amino acids on the AA sensitivity of
hIK1. A representative experiment for the V275A-hIK1 construct is shown
in Fig. 5E. Although AA significantly inhibited 43 ± 11% (315 ± 81 pA and 165 ± 37 pA in the absence and
presence of AA, respectively, n = 9; p < 0.05) of the V275A current (Fig. 5F) this inhibition is
significantly less than the 82 ± 2% inhibition observed for wild
type hIK1 (p < 0.05). These data suggest that
Val275 is also contributing to the inhibition of hIK1 by
AA.
Lastly, we predicted that the double mutated T250S/V275A-hIK1 channel
would be relatively insensitive to AA. Similar to the T250S channel
construct, the double-mutated channel was not sensitive to AA, with
current averaging 191 ± 39 pA and 192 ± 47 pA
(n = 4) in the absence and presence of AA, respectively
(Fig. 5F). Clearly, these data suggest that the amino acids
Thr250 and Val275 are critical in conferring
the AA sensitivity of hIK1.
Can the Thr250 and Val275 Substitutions
Confer AA Sensitivity to rSK2?--
If Thr250 and
Val275 are crucial for AA inhibition of hIK1, then we
hypothesized that introducing these amino acids at the appropriate amino acid alignment positions in rSK2 would cause rSK2 to become sensitive to AA inhibition. Thus, we made separate rSK2 constructs in
which Ser359 was mutated to threonine (S359T-rSK2),
Ala384 was mutated to valine (A384V-rSK2), or the double
mutation S359T/A384V-rSK2. A representative experiment of the
S359T-rSK2 channel is shown in Fig.
6A. The average channel
current prior to the addition of AA was 277 ± 27 pA, which was
reduced (p < 0.001) to 223 ± 30 pA in the
presence of AA, indicative of a 21 ± 3% inhibition of channel
current (Fig. 6D, n = 4). This inhibition is
significantly greater than the 8 ± 3% inhibition observed for
wild-type rSK2 (Fig. 2), indicating that the S359T mutation confers a
partial AA binding site to rSK2. Similarly, the A384V-rSK2 mutation
induced a partial sensitivity to AA (Fig. 6B). In excised,
inside-out patch-clamp experiments, A384V-rSK2 current was inhibited an
average of 31 ± 3% (Fig. 6D), from 241 ± 61 pA
to 172 ± 55 pA in the presence of AA (n = 3;
p < 0.01). Again, this inhibition is significantly greater than that observed for wild-type rSK2 (p < 0.05), indicative of the fact that a partial AA binding site can be
introduced into rSK2 with this mutation.

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Fig. 6.
Thr250 and Val275
substitutions into rSK2 (S359T and A384V) confer AA sensitivity.
Representative current (pA) traces of the mutated rSK2 channels with
S359T (A), A384V (B), or the double mutation
S359T/A384V (C) in response to 3 µM AA.
D, percentage inhibition summary data for all experiments
for the mutated channels are given (number of experiments shown above
the bar). Data for rSK2 are also presented for comparison.
Mutated rSK2 channels were heterologously expressed in HEK cells and
recorded in excised inside-out patches at a holding potential of 100
mV. 0 Ca2+ steps occurred at the end of the experiments to
illustrate the Ca2+ dependence of the mutated
channels.
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Based on the above observations that both S359T and A384V induced a
partial sensitivity for AA inhibition on rSK2 we determined whether the
double mutation, S359T/A384V-rSK2, would create an rSK2 channel whose
sensitivity to AA was similar to the sensitivity observed for hIK1. A
representative experiment for this double mutant is shown in Fig.
6C. Arachidonic acid reduced (p < 0.001) the channel current of S359T/A384V-rSK2 an average of 63 ± 8% (Fig. 6D) from 240 ± 34 pA in the absence of AA to
73 ± 14 pA in the presence of AA (n = 9). These
data clearly demonstrate that Thr250 and Val275
are critical for AA sensitivity of hIK1 and that similar amino acid
substitutions confer AA sensitivity to rSK2, a channel which is
normally insensitive to AA.
Arachidonic Acid and Clotrimazole Share Overlapping Inhibitory
Sites--
As noted above, our results suggest that AA and
clotrimazole share similar molecular sites of interaction, resulting in
block of hIK1. That is, Chandy and co-workers (36) previously
demonstrated that T250 and V275 in hIK1 were critical for inhibition by
clotrimazole. Similarly, we demonstrate that these amino acids play a
crucial role in the inhibition of hIK1 by AA. Indeed, substituting
these amino acids into their corresponding positions in rSK2
(S359T/A384V) results in the generation of an AA-sensitive rSK2
channel. To confirm the extent of molecular overlap between the AA and
clotrimazole inhibitory sites we similarly evaluated the sensitivity of
hIK1, rSK2, and the double mutations hIK1-T250S/V275A and
rSK2-S359T/A384V to 3 µM clotrimazole. As shown for one
representative experiment in Fig.
7A, clotrimazole dramatically
inhibited hIK1, similar to what has been previously reported (27, 36,
38, 39). In five experiments, clotrimazole inhibited hIK1 an average of 94 ± 3%, from 379 ± 171 pA to 17 ± 7 pA
(p < 0.01, Fig. 7E). In contrast, in
patches containing rSK2 the current averaged 571 ± 279 pA, and
this was not inhibited by clotrimazole (540 ± 262 pA; 3 ± 2%, n = 6; Fig. 7, B and E), as
previously reported (36). Similar to what was reported by Chandy and
co-workers (36), we demonstrate that the effect of clotrimazole on
hIK1-T250S/V275A is significantly reduced compared with wild-type hIK1
(p < 0.01, Fig. 7C). That is, on average
clotrimazole reduced (p < 0.01) the current an average
of only 31 ± 8%, from 64 ± 30 pA to 45 ± 25 pA
(n = 5, Fig. 7E). Finally, as shown in Fig.
7, D and E, we demonstrate that mutating
Ser359 and Ala384 in rSK2 to their
corresponding amino acids in hIK1 (rSK2-S359T/A384V) confers
clotrimazole sensitivity onto rSK2. In six patches, clotrimazole inhibited 92 ± 3% of current flow across patches expressing
rSK2-S359T/A384V from an average of 80 ± 37 pA to 3 ± 1 pA
(p < 0.01). These results confirm previous studies
(36) and further demonstrate that identical pore lining amino acids are
required to confer both AA- and clotrimazole-dependent inhibition of hIK1.

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Fig. 7.
Effect of clotrimazole on hIK1, rSK2,
hIK1-T250S/V275A, and rSK2-S359T/A384V. Representative current
(pA) traces for hIK1 (A), rSK2 (B),
hIK1-T250S/V275A (C), and rSK2-S359T/A384V (D) in
response to 3 µM clotrimazole. E, percentage
inhibition summary data for all experiments are given (number of
experiments shown above the bars). All channels were
heterologously expressed in HEK cells and recorded in excised,
inside-out patches at a holding potential of 100 mV. 0 Ca2+ steps occurred at the end of the experiments to
illustrate the Ca2+ dependence of these channels.
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DISCUSSION |
Arachidonic acid is an ubiquitous second messenger released in
response to both Ca2+-and cAMP-mediated agonists, including
acetylcholine (40, 41), UTP (42-45), bile acids (17), adenosine (16),
and vasoactive intestinal polypeptide (14). Inflammatory responses are
also characterized by significant increases in AA production, both via
direct release from inflammatory cells (20) as well as through Ca2+-dependent, kinin-mediated generation (18).
Given the crucial role of ion channels in generating the physiological
as well as patho-physiological responses to these agonists it is not
surprising that K+, Na+, Ca2+, and
Cl
channels have all been demonstrated to be modulated by
either AA directly or one of its oxygenated metabolites (22, 23). In
this regard, we previously demonstrated that hIK1, endogenously expressed in the colonic cell line T84, was inhibited with high affinity (IC50 = 0.42 µM) by AA (2). This
inhibitory action appeared to be a direct effect on hIK1, as inhibitors
of COX, LOX, or cytochrome P-450 did not modulate this
AA-dependent inhibition (2).
In the present study, we confirm that AA inhibits hIK1, heterologously
expressed in HEK293 cells, with high affinity (IC50 = 1.4 ± 0.7 µM; Fig. 1C), a value very
similar to what we previously reported for endogenously expressed hIK1
(2). Consistent with our previous studies, we demonstrate that ETYA, a
blocker of COX, LOX, and P450 pathways (32, 33) did not alter the
inhibitory effect of AA on hIK1 (Fig. 1D), further
supporting our conclusion that the effects of AA on hIK1 are direct. We
further demonstrate that AA does not inhibit the activity of rSK2,
another member of the KCNN gene family. As we previously
demonstrated that hIK1 is activated by ATP/PKA while SK1-3 are not
(31), the present results further highlight the unique regulatory
properties of hIK1 relative to the SK channels.
Given the high degree of homology between IK and SK channels
throughout their core regions (S1-S6) we anticipated that the inhibitory action observed would be directed against either the cytoplasmic NH2 or COOH terminus. Indeed, three different
NH2-terminal splice variants of ROMK exist, only one of
which is sensitive to AA (34). Interestingly, AA induces the
phosphorylation of ROMK at a non-consensus NH2-terminal
phosphorylation site (34). In contrast to these results, we demonstrate
that the cytoplasmic NH2-terminus of hIK1 does not play a
role in the AA-dependent inhibition of hIK1 (Fig.
3A). Also, the molecular site for AA-dependent activation of TREK-2 has previously been mapped to the proximal COOH
terminus (35). We demonstrate that, in contrast to these results, the
cytoplasmic COOH terminus of hIK1 does not play a role in the
AA-dependent inhibition observed (Fig. 3B).
Mutational analysis demonstrates that AA interacts with the pore of
hIK1. Indeed, while the T250S mutation completely abrogated the effect
of AA, we further demonstrate that Val275, an amino acid in
S6 predicted to line the pore, also plays a crucial role in the
AA-dependent inhibition of hIK1. As the side chains of
Thr250 and Val275 are predicted to extend into
the water-filled pore of hIK1 (36) our results would most
parsimoniously be interpreted as demonstrating a direct pore block of
hIK1 by AA. Somewhat surprisingly, these exact amino acids were
previously demonstrated to be required for the inhibition of hIK1 by
clotrimazole (36). Indeed, our own studies have confirmed the role of
Thr250 and Val275 in the inhibition of hIK1 by
clotrimazole. Thus, our results indicate that clotrimazole and its
analogues have been synthesized to take advantage of an already
pre-existing second-messenger binding site (AA), which similarly blocks
current flow through the pore of hIK1. Clearly, the most convincing
argument in favor of Thr250 and Val275 defining
the binding site for AA is our demonstrated ability to recapitulate
this binding site in a closely related but AA-insensitive channel, rSK2
(Fig. 6). Indeed, while mutation of Thr250 alone in hIK1
was sufficient to completely eliminate the inhibition by AA,
introducing this single point mutation into rSK2 (S359T) conferred only
partial inhibition by AA (Fig. 6A). Similarly, the A384V
mutation resulted in only a partial block of rSK2 in response to AA
(Fig. 6B). However, mutation of both amino acids resulted in
a rSK2 channel that was nearly as sensitive to AA as hIK1 (Fig.
6C). In total, our results suggest that the molecular motif
required for both AA and clotrimazole inhibition of hIK1 share a high
degree of overlap and may indeed be identical.
It is interesting to note that both clotrimazole- (36) and
AA-dependent inhibition of hIK1 is highly dependent upon a
threonine in the pore, as this threonine is conserved in virtually all
K+ channels, including voltage-gated (e.g.
Kv1-4), inward rectifying (e.g. ROMK, KATP, GIRK), and two
pore-domain (e.g. TWIK, TREK, TRAAK) K+
channels. Indeed, AA is known to both inhibit and activate a wide array
of K+ channels, all of which share this pore threonine.
Thus, the specificity of actions is likely conferred by additional
amino acids, including Val275 in S6. Indeed, we demonstrate
that a single S/T mutation in rSK2 confers only modest sensitivity to
inhibition (21%) by AA (Fig. 6). While we have not evaluated the
contribution of all of the pore-lining amino acids on AA sensitivity,
other amino acids may also modulate AA affinity, perhaps explaining the
wide range of affinities reported for AA-dependent
modulation of K+ channels (22, 23). Based on our studies,
it would be predicted that channels sharing both a threonine
corresponding to Thr250 in hIK1 and valine corresponding to
Val275 in hIK1 would be sensitive to AA. This is indeed the
case in many instances, including Kv1.1 (46), Kv1.3 (47), Kv1.5 (48), and Kv4.2 (49), although the K+ channel-interacting protein
KChIP1 modifies this effect on Kv4.2 (32). Future studies will be
required to determine whether the conserved, pore-lining threonine and
valine are responsible for the inhibition of these channels by
arachidonic acid. However, in other channels sharing both this
threonine and valine, including Kir2.1 and the Maxi (BKCa)
K+ channel, AA has been shown to have either no effect
(Kir2.1, Ref. 33) or is stimulatory (BKCa, Refs. 50-52).
Importantly, Chandy and co-workers (53) previously demonstrated that
Kv1.1, Kv1.3, Kv1.5, Kv4.2, Kir2.1, and BKCa channels were
also insensitive to clotrimazole despite the conservation of the
threonine and valine. As previously suggested (53), this may indicate
that the orientation of the threonine and valine side chains are
altered by the surrounding amino acids resulting in widely disparate
sensitivities to both clotrimazole and AA despite an apparently
conserved binding site.
In conclusion, we demonstrate that the inhibition of hIK1 by AA is
dependent upon two critical pore lining amino acids, Thr250
and Val275. As the pore of the related channels, SK1-3
would be predicted to be slightly wider at this binding site due to the
shorter amino acid side-chain lengths of serine and alanine relative to
threonine and valine, AA fails to interact with and block SK2. Our
results further clarify the mechanism by which the ubiquitous second
messenger, AA modulates physiologic functions via the inhibition of
these Ca2+-dependent K+ channels.
Interestingly, the molecular mechanisms of AA action have been mapped
on three separate classes of K+ channels, the Kir
(ROMK) (34), two-pore (TREK-2) (35), and now six-transmembrane,
Ca2+-dependent channels; each of these channels
exhibits a unique mechanism of action for AA. As AA modulates the
activity of numerous other K+ channels it will be
enlightening to determine whether additional motifs are delineated or
whether some conservation of molecular action begins to emerge.