From the Department of Pharmacology University of Washington, Seattle, Washington 98195-7280
Received for publication, November 6, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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We previously showed that activation of the human
endothelin A receptor (HETAR) by endothelin-1 (Et-1) selectively
inhibits the response to mu opioid receptor (MOR) activation of the
G-protein-gated inwardly rectifying potassium channel (Kir3). The Et-1
effect resulted from PLA2 production of an eicosanoid that inhibited Kir3. In this study, we show that Kir3 inhibition by eicosanoids is
channel subunit-specific, and we identify the site within the channel
required for arachidonic acid sensitivity. Activation of the
G-protein-coupled MOR by the selective opioid agonist
D-Ala2Glyol, enkephalin, released G The G-protein-gated inwardly rectifying potassium channels
(Kir3)1 provide essential
regulation of neuronal and cardiac excitability (1). By mediating the
effects of acetylcholine, monoamine, and peptide receptor activation,
Kir3 channels respond to a wide range of transmitters. The Kir3 family
of G-protein-gated inwardly rectifying potassium channels consists of
subunits (Kir3.1-Kir3.5) that can assemble to form functional
heteromultimers. Kir3 channels share a common design characterized by
cytoplasmic N and C termini and two transmembrane domains M1 and M2
that surround a potassium selective pore region (P or H5) (2) (see Fig.
1 below). The activation of Kir3 is
complex and not completely understood. Kir3 channels require the
interaction of multiple components to produce channel activation,
including G that
activated Kir3. The response to MOR activation was significantly
inhibited by Et-1 activation of HETAR in homomeric channels composed of
either Kir3.2 or Kir3.4. In contrast, homomeric channels of Kir3.1 were
substantially less sensitive. Domain deletion and channel chimera
studies suggested that the sites within the channel required for
Et-1-induced inhibition were within the region responsible for channel
gating. Mutation of a single amino acid in the homomeric Kir3.1 to
produce Kir3.1(F137S)(N217D) dramatically increased the channel
sensitivity to arachidonic acid and Et-1 treatment. Complementary
mutation of the equivalent amino acid in Kir3.4 to produce
Kir3.4(S143T)(D223N) significantly reduced the sensitivity of the
channel to arachidonic acid- and Et-1-induced inhibition. The critical
aspartate residue required for eicosanoid sensitivity is the same
residue required for Na+ regulation of PIP2
gating. The results suggest a model of Kir3 gating that incorporates a
series of regulatory steps, including G
, PIP2,
Na+, and arachidonic acid binding to the channel gating domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, Na+, the phospholipid PIP2,
and ATP (3-7). Huang and colleagues proposed that G
activates
Kir3 by stabilizing interactions between PIP2 and the
potassium channel. They showed that PIP2 depletion blocks
activation of Kir3 by both G
and Na+ (8). Both the
stimulation and inhibition of Kir3 mediated by G
were recently
shown to depend on the type of
subunit in the dimer (9). ATP is
another regulatory element in Kir3 gating that presumably acts
indirectly by maintaining phosphorylation of PIP2. The
response to activation of Kir3.1 and Kir3.4 heteromultimers is rapidly
inactivated (run down) when internal ATP is depleted. Sui et
al. (10) show that Kir3 activity is ATP-dependent and is mediated by PIP2; moreover, ATP hydrolysis enables both
Na+ and G
activation (10). These studies support the
hypothesis that Kir3 gating components responsible for channel
activation are interdependent.
View larger version (24K):
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Fig. 1.
Diagram of Kir3. Kir3 channels share a
common design characterized by a cytoplasmic N terminus (~90 amino
acids) and C terminus with two transmembrane domains M1 and M2, which
surround the ion-selective pore region (P or H5). Kir3 channels
function as active heteromultimers (e.g. Kir3.1 pairs with
other subtypes). Mutations in the P region enhance the activity of
homomers. This diagram shows the P region with the site of specific
point mutations that produce functional homomeric channels:
Kir3.1(F137S), Kir3.2(S146T), and Kir3.4(S143T). The presumed
PIP2-Na+-G interaction site in the C
terminus is indicated. Kir3 is also inhibited by tyrosine
phosphorylation (11) with phosphorylation sites in the N terminus noted
(
). Other sites of G
interaction have been identified, but are
not illustrated.
Kir3 normally exists as a heterotetramer, but the gating properties of individual subunits may be studied by using channel mutants able to form functional homomeric channels: Kir3.1(F137S) (11), Kir3.2(S146T) (12), and Kir3.4(S143T) (13). Although members of Kir3 are similar in structure, one primary difference in the subunits is the presence of a Na+ activation site found in Kir3.2 and Kir3.4, which is not found in Kir3.1. Ho and Murrell-Lagnado (14, 15) used chimeras of the Na+-insensitive Kir3.1 and the Na+-sensitive Kir3.2 to define the site on the channel that was sensitive to Na+ activation. Substitution of asparagine for aspartate 226 in Kir3.2(D226N) abolished Na+-dependent activation for both the Kir3.2 homomer and Kir3.1/Kir3.2 heteromultimers without altering the amplitude of receptor activation (14, 15). Ho and Murrell-Lagnado proposed that Na+ binding to an aspartic acid residue in Kir3.2 masks a nearby charged amino acid and permits PIP2 binding.
In contrast to the process of channel activation by PIP2,
Na+, and G, the mechanism of arachidonic acid
inhibition of Kir3 is less well defined. Arachidonic acid and its
metabolites modulate several ion channels, including Kir3.1 and Kir3.4
heteromultimers in cardiac myocytes (16). Unsaturated free fatty acids
such as oleic, linoleic, and arachidonic acids inhibit Kir3.1 and
Kir3.4 heteromultimers by blocking ATP-dependent gating in
atrial cells (7). We previously showed that arachidonic acid inhibits
the potassium channel response to the mu opioid agonist DAMGO in
Xenopus oocytes expressing MOR and Kir3 heteromultimers
(17). Channel sensitivity to arachidonic acid depended on the channel
subtype: heteromultimers consisting of Kir3.1 and Kir3.2 or Kir3.1 and Kir3.4 were more sensitive to arachidonic acid than heteromultimers consisting of Kir3.1 and Kir3.5. These results suggest that eicosanoids have direct effects on G-protein-gated inwardly rectifying potassium channels by modification of the channel conformation, but the molecular
basis for eicosanoid inhibition of Kir3 was not defined. In the present
study, we explored the hypothesis that eicosanoids generated by HETA
activation may directly modulate Kir3 gating. Using site-directed
mutagenesis to identify the eicosanoid-sensitive regulatory site on the
Kir3 channel, we provide evidence that eicosanoid-induced inhibition of
Kir3 requires the Na+-dependent gating site.
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EXPERIMENTAL PROCEDURES |
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Complementary DNA Clones and cRNA Synthesis-- The rat mu opioid receptor clone was obtained from Dr. Lei Yu (GenBankTM accession number L13069). cDNA for the human endothelin A (HETA) receptor (GenBankTM accession number S67127) was obtained from Dr. Richard Kris. cDNAs for the Kir 3.1 (GIRK1) (GenBankTM accession number U01071) and Kir 3.2 (GIRK2) (GenBankTM accession number U11859) were obtained from Drs. Cesar Lebarca and Henry Lester. Dr. John Adelman provided the Kir 3.4 (GIRK4) clone (GenBankTM accession number X83584). Kir3 chimeras were the kind gift of Dr. Diomedes Logothetis. Point mutations to produce functional homomeric channels; Kir3.1(F137S) (11), Kir3.2(S146T) (12), and Kir3.4(S143T) (13) were constructed. Mutations were introduced by polymerase chain reaction amplification using Pfu turbo DNA polymerase with complementary oligonucleotide primers incorporating the desired mutation. Positive clones were confirmed by automated sequencing. Plasmid templates for constructs were linearized prior to in vitro cRNA synthesis using mMESSAGE mMACHINE (Ambion Inc., TX).
Oocyte Maintenance and Injection-- Healthy stage V and VI oocytes were harvested from mature anesthetized Xenopus laevis (Nasco, Ft. Atkinson, WI) and defolliculated enzymatically as described previously (18). The oocytes were maintained at 18 °C in standard oocyte buffer, ND96 (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.5) supplemented with 2.5 mM sodium pyruvate and 50 µg/ml gentamicin (Sigma Chemical Co.). One day after harvest, each oocyte was injected with 50 nl of cRNA for the mu opioid receptor (MOR), human endothelin A receptor (HETA), and G-protein inwardly rectifying potassium channels. Recordings were made at least 48 h after cRNA injection.
Electrophysiological Studies--
A Geneclamp 500 amplifier was
used for standard two-electrode voltage-clamp experiments. The pCLAMP
program (Axon Instruments) was used for data acquisition and analysis.
Oocytes were removed from incubation medium, placed in the recording
chamber containing ND96 medium, and clamped at 80 mV. Recordings were
made in hK buffer (2 mM NaCl, 96 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.5). Microelectrodes were filled with 3 M KCl and had resistances of 0.5-1.0 M
. Currents were
measured without leak subtraction. Individual comparisons of drug
effects on Kir3 were conducted using oocytes from the same harvest and
injection batch. Pharmacologic agents were perfused or placed directly
into the bath from freshly made stock solutions.
Materials--
Stock solutions of arachidonic acid were
dissolved in dimethyl sulfoxide (Me2SO); the final
concentration of Me2SO applied to the oocytes was <0.02%.
Arachidonic acid was stored at 70 °C until use. Nitrogen was
bubbled through water prior to dissolving endothelin. Endothelin-1 and
DAMGO were obtained from Phoenix Pharmaceuticals, Belmont, CA and were
stored at
20 °C until use. Arachidonic acid and U73122 were from
Calbiochem, La Jolla, CA. Me2SO was from Sigma Chemical
Co., St. Louis, MO.
Statistical Analysis--
Data are presented as means ± S.E. The statistical significance of differences between results was
calculated using ANOVA followed by a student's unpaired two-tailed
t test. A probability of p < 0.05 was
considered statistically significant.
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RESULTS |
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Effects of Endothelin-1 on Kir3 Activation by
MOR--
Heterologous expression of MOR and Kir3 in Xenopus
oocytes generated a malleable system for the study of channel
activation (Fig. 2). Activation of the
Gi/Go-coupled opioid receptors by the
agonist DAMGO (1 µM) released G that activated Kir3
and produced a robust inward current (Fig.
2A). Activation of the heterologously expressed Gq-coupled endothelin receptor
produced a cascade of signals that resulted in the reduction of the
DAMGO-evoked response.
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To identify the Kir3 subunit conferring sensitivity to endothelin, we
used the strategy developed by Logothetis and colleagues to generate
functional homomeric Kir3 channels. Mutation in the pore of these
channels, Kir3.1(F137S) and Kir3.4(S143T) greatly increased expression
and the current evoked by the channel homomers. In agreement with
Vivaudou and colleagues (13), we noted that the wild type channel
Kir3.4 does not produce functional currents when expressed alone in
Xenopus oocytes. In contrast, the homomer Kir3.4(S143T) (1 ng of cRNA/oocyte) produced a robust current with an average
receptor-activated current of 1-2 µA in response to agonist.
Although the wild type channel Kir3.1 (1 ng) forms functional channels
with the intrinsic Xenopus subunit Kir3.5, the current
response produced was small (50-100 nA) compared with the large
currents (1-2 µA) produced by the functional homomer Kir3.1(F137S)
(1 ng). These results are consistent with the conclusions of Vivaudou
et al. (13) that the Kir3.1 and Kir3.4 currents are produced
by the functional homomeric channel without combining with endogenous
Kir3.5. Similarly, Kir3.2(S146T) (1 ng) produced larger currents (500 nA to 1 µA) compared with wild type Kir3.2 (100 nA) (1 ng), which
normally forms a homomeric channel. Moreover, the point mutation of
Kir3.2(S146T) served as a control as this mutation in the pore was
similar to Kir3.1(F137S) and Kir3.4(S143T). Thus, oocytes expressing
presumptive homomeric forms of Kir3.1, 3.2, and 3.4 were functional and
were robustly activated by G released by mu opioid receptor activation.
Oocytes expressing either Kir3.2(S146T) or Kir3.4(S143T) treated with Et-1 prior to the second DAMGO challenge showed a marked inhibition of the second opioid response (Fig. 2B). The amplitude of the second opioid response in Kir3.2(S146T) homomers after Et-1 treatment was inhibited by 100 ± 0% (n = 9). The amplitude of the second opioid response in Kir3.4(S143T) homomers after Et-1 treatment was inhibited by 74 ± 5% (n = 18) (p < 0.01) (Fig. 2B). In contrast, the second opioid response in oocytes injected with Kir3.1(F137S) and treated with Et-1 prior to the second DAMGO challenge was not significantly inhibited. The amplitude of the second opioid response in Kir3.1(F137S) homomers after Et-1 treatment was 1 ± 11% (n = 16). The difference in endothelin sensitivity of the Kir3.1-3.5 heteromeric channel and the Kir3.1(F137S) channel supports the conclusion that the latter formed a functional homomer under these expression conditions. In addition, the channel type selectivity evident from this experiment further supports the conclusion that the endothelin-induced suppression of the DAMGO-activated response was caused by a direct modification of the channel.
We previously showed that the PLA2 inhibitor AACOCF3 selectively blocked the endothelin receptor-mediated effect (17). These results suggested that an eicosanoid such as arachidonic acid produced by PLA2 activation following HETA receptor activation was responsible for channel inhibition. Another interpretation of our data was that the endothelin effect was caused by PLC-mediated PIP2 depletion. To test the latter hypothesis, oocytes were pretreated with the PLC inhibitor U73122 (5 µM) (19) for 10 min and tested for endothelin sensitivity under conditions previously shown to inhibit PLC activity in oocytes (20). The amplitude of the second opioid response in Kir3.4(S143T) homomers after Et-1 treatment was inhibited by 61 ± 5% (n = 8). Pretreatment with the PLC inhibitor U73122 (5 µM) did not block the inhibition of the second opioid response to Et-1. The amplitude of the second opioid response of Kir3.4(S143T) homomers after Et-1 treatment was inhibited by 66 ± 5% (n = 8) (not significantly different than in the absence of U73122). Thus, PLA2 activation and subsequent eicosanoid production likely caused the endothelin-induced inhibition. Furthermore, the results suggest that Kir3.2 and Kir3.4 were sensitive to the eicosanoid generated by PLA2 activation whereas Kir3.1 was not.
Endothelin Effects on Kir3 Channel Truncations and
Chimeras--
To determine the basis for the insensitivity of
Kir3.1(F137S) to endothelin receptor activation, the Et-1-insensitive
Kir3.1 and Et-1-sensitive Kir3.4 sequences were aligned and compared. These Kir3 subunits share the most homology in the pore region and the
greatest heterogeneity in the tail regions. Macica and colleagues (21,
22) identified a serine residue in the distal N terminus in the inward
rectifier ROMK1 that confers sensitivity to arachidonic acid. We wanted
to test the hypothesis that an N-terminal residue was responsible for
eicosanoid sensitivity of Kir3. Sequential truncations of the N
terminus were made, and the effect of Et-1 on Kir3 activation was
tested. Oocytes expressing a truncated Kir3.4(S143T) lacking amino
acids 1-57 produced strong potassium currents and a robust response
following DAMGO activation of MOR. The amplitude of the second opioid
response in the channel with the N-terminal truncation
Kir3.4(S143T)(1-57) after Et-1 treatment was inhibited, a result
not significantly different than the parent (p > 0.05)
(Fig. 3B). Truncation of the
first 23 amino acids in Kir3.2 also did not block either the DAMGO
activation or endothelin sensitivity (data not shown).
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In contrast, the C terminus of Kir3.1 and Kir3.4 differed significantly in both amino acid length and amino acid sequence. Chimeras composed of the Et-1-sensitive Kir3.4(S143T) N terminus and pore region with the Et-1-insensitive Kir3.1 C-terminal tail, Kir3.4(S143T)-(1-338)/Kir3.1-(333-501) and Kir3.4(S143T)-(1-249)/Kir3.1-(244-501), were used to define the region of endothelin sensitivity. The response to DAMGO of oocytes expressing the channel chimera Kir3.4(S143T)-(1-338)/Kir3.1-(333-501) was inhibited following Et-1 treatment. The sensitivity of the chimera was not significantly different from the parent channel Kir3.4(S143T) (p > 0.05) (Fig. 3B). Similarly, the DAMGO response of oocytes expressing the chimera Kir3.4(S143T)-(1-249)/Kir3.1-(244-501) was inhibited following Et-1 treatment. The sensitivity of this chimera was also not significantly different from Kir3.4(S143T) (p > 0.05) (Fig. 3B). Thus, neither the N terminus nor the distal C terminus of Kir3 contained elements required for the Et-1-induced inhibition. The chimera and truncation data suggest that the channel domain responsible for endothelin sensitivity was within the proximal C-terminal region near the transmembrane domain M2.
The proximal C-terminal domain of the channel contains sites
responsible for G, PIP2, and Na+ gating
(5, 9, 14, 15). After comparing the different Kir3 channels, we noted a
correlation between endothelin sensitivity and Na+ gating
sensitivity. Kir3.1 was substantially less sensitive to endothelin,
whereas Kir3.2 and Kir3.4 were very sensitive (Fig. 2B).
Moreover, Kir3.1 is not gated by Na+, whereas Kir3.2 and
Kir3.4 are gated by Na+ (5, 14, 15). Based on this
correlation, we explored the hypothesis that the Kir3 gating domain was
important in the observed eicosanoid inhibition of Kir3.
Na+ sensitivity was reported to depend on the presence of a
critical aspartate residue in the gating domain (5, 14, 15).
Kir3.1(F137S) and Kir3.4(S143T)(D223N) lack the aspartate thought to be
responsible for Na+ gating, and the corresponding channels
Kir3.4(S143T) and Kir3.1(F137S)(N217D) contain this residue. These
channel variants were produced and expressed in oocytes (Fig.
4).
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The Effect of the Sodium Binding Site on the Endothelin-1 Inhibition of Kir3-- We coexpressed either Na+-sensitive or Na+-insensitive channel mutants of Kir3.1 or Kir3.4 with cRNA for the MOR and cRNA for the human endothelin A receptor. The amplitude of the second opioid response in Kir3.1(F137S) (Na+-insensitive) homomers after Et-1 treatment was inhibited by 22 ± 5% (n = 18) (Fig. 4). The slightly greater sensitivity to Et-1 shown by Kir3.1(F137S) in this set of recordings compared with the data presented in Fig. 2 was not statistically different from the data shown in Fig. 1. The slight difference is attributed to normal seasonal variation in oocytes, and the increased sensitivity to Et-1 was also shown by Kir3.4(S143T). Introduction of the aspartate residue that confers Na+ sensitivity also dramatically increased endothelin sensitivity. Oocytes injected with Kir3.1(F137S)(N217D) (Na+-sensitive) and then treated with Et-1 prior to the second DAMGO challenge showed a marked inhibition of the second opioid response. The second opioid response after Et-1 treatment was inhibited by 82 ± 5% (n = 16) (p < 0.01).
For Kir3.4, removal of the critical aspartate significantly reduced endothelin sensitivity (Fig. 4). The second mu opioid response after Et-1 activation in oocytes expressing the HETA receptor, the MOR and Kir3.4(S143T) (Na+-sensitive) was inhibited by 97 ± 1% (n = 7). Oocytes expressing Kir3.4(S143T)(D223N) (Na+-insensitive) treated with Et-1 prior to the second DAMGO challenge showed significantly less inhibition of the second opioid response; endothelin reduced the response to DAMGO by 63 ± 6% (n = 12). Kir3.4(S143T)(D223N) remains partially sensitive to endothelin treatment, but the sensitivity was significantly reduced compared with Kir3.4(S143T). These data indicate that a significant component of the endothelin sensitivity of Kir3 depended on the presence of the aspartate residue also responsible for Na+ gating. The residual sensitivity suggests that endothelin receptor activation may also inhibit Kir3 at other sites.
Arachidonic Acid Effects on Channel Homomers--
The
results suggested that endothelin activation of PLA2 produced an
eicosanoid that inhibited Kir3 channels by interacting at the
Na+ gating domain. Prior work showed that arachidonic acid
was the most potent of the eicosanoids at inhibiting Kir3 in cardiac
myocytes (7). Based on this finding, we tested the effectiveness of arachidonic acid application on the Na+-sensitive and
-insensitive Kir3 variants. In oocytes expressing the channel homomer
Kir3.1(F137S), arachidonic acid (20 µM) did not inhibit
the second DAMGO-elicited response of MOR (0 ± 3%). In oocytes
expressing the channel with a site for Na+ activation,
Kir3.1(F137S)(N217D), arachidonic acid produced an inhibition of the
second opioid response; the second DAMGO response was inhibited by
(26 ± 1%) (p < 0.05) (Fig.
5). Moreover, arachidonic acid inhibited
the Na+-sensitive Kir3.4(S143T) (18 ± 2%). compared
with oocytes with the analogous Na+-site removed
Kir3.4(143T)(D223N) (2 ± 6%). These data support the
hypothesis that eicosanoid inhibition of Kir3 was dependent on the
presence of the Na+ gating site.
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DISCUSSION |
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The principal finding of this study is that arachidonic acid
inhibited G-protein-gated potassium channels at the
Na+/PIP2 gating domain. The identification of
this mechanism helps clarify the effects of eicosanoids generated by
phospholipase A2 activation, and the results provide new insights into
the gating process controlling Kir3 function. Previous studies
established that the elevation of G concentration following
Gi/o-coupled receptor activation increases Kir3 conductance
(23). G
was shown to work in concert with PIP2 to
induce a conformational change in the channel and open the
K+-selective pore. In addition to G
, Kir3.2,
and Kir3.4 channel subunits are activated by Na+ (14, 15).
The results provide support for the hypothesis that arachidonic acid is
an additional component, directly regulating Kir3 gating.
The results obtained provide evidence that the production of an
eicosanoid by endothelin receptor activation was responsible for the
observed inhibition of Kir3 in this expression system. The HETAR is a
Gq-coupled receptor that activates both PLC and PLA2 (24).
As PLA2 metabolizes PIP2 to arachidonic acid (Fig. 6), depletion of PIP2 could
potentially result from endothelin receptor activation. Either
mechanism could result in Kir3 channel inhibition, because the
production of an eicosanoid (7) or the depletion of PIP2
(20) could reduce channel conductance. Meyer et al. (25)
recently showed that endothelin receptor activation inhibits IK(ACh) in
atrial myocytes, and this effect could be blocked by increasing the
intracellular concentration of PIP2. They interpret their
data to suggest that endothelin receptor activation of PLC results in
PIP2 depletion and IK(ACh) inhibition; however the role of
an eicosanoid in the endothelin effect was not considered and
competition between activation by PIP2 and inhibition by
arachidonic acid could explain their result (Fig. 6).
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Several lines of evidence exclude the explanation that depletion of PIP2 was responsible for the endothelin effect in oocytes. We found that PLC inhibition by U73122 did not block endothelin action. Our prior study showed that the PLA2 inhibitor AACOCF3 blocked endothelin effects (17). The third observation is that the Kir3 subunit sensitivity to arachidonic acid matches endothelin sensitivity. Thus, our results support the conclusion that endothelin activation of PLA2 produces an eicosanoid that inhibits Kir3 rather than depletion of PIP2 required for channel activation. The specific eicosanoid generated by endothelin receptor activation in Xenopus oocytes was not identified; however, arachidonic acid is a reasonable candidate based on its high potency shown by Kim and Pleumsamran (7). In addition, arachidonic acid exhibited an inhibition profile that was similar to that endothelin receptor activation (Fig. 5).
We note that Kir3.4(S143T)(D223N) was not significantly affected by arachidonic acid application whereas Kir3.4(S143T)(D223N) had residual sensitivity to endothelin inhibition. The difference could either result from the possibility that endothelin acted by additional mechanisms or that the activation of the HETAR was a more efficient means to deliver the active eicosanoid than extracellular application of arachidonic acid. Consistent with the latter interpretation, the data showed that the inhibition produced by endothelin was more robust than that produced by arachidonic acid. These results suggest that the critical aspartate residue was not the sole determinant of endothelin effect. Additional site-directed mutagenesis would be required to define other residues in the binding pocket required for eicosanoid sensitivity.
The basis for the inhibitory effects of eicosanoids on Kir3
conductance was not defined, but the study provides clues. The correlation between Na+ sensitivity and arachidonic acid
sensitivity suggests that the binding site of eicosanoids is part of
the domain controlled by the Na+ binding site. Moreover,
the Na+ binding site also regulates PIP2
binding. Because arachidonic acid shares structural features with
PIP2, the PIP2 binding region of Kir3 may also
bind arachidonic acid. The inhibition of gating caused by arachidonic
acid may result from a competition with PIP2 for binding at
this site. In the absence of structural data, we suggest the physical
basis for the interaction between these gating components and the ion
channel in a diagram (Fig. 6). Lei et al. (9) used mammalian
cells transfected with Kir3 heterotetramers to show that
G5-containing dimers could inhibit Kir3. They suggested that
G
5-containing dimers could competitively displace G
binding in
the proximal C terminus, a region near the putative eicosanoid binding
site suggested in by the present study. Additional work will be
required to further define the interaction between eicosanoids, G
, and PIP2 at Kir3; however, our data show that the
mechanism of Kir3 sensitivity to arachidonic acid differs from that
reported for ROMK channels by Macica et al. (21, 22).
The large Kir3.1(F137S) currents clearly showed that the channel was formed as a homomer consistent with prior reports (11, 13). The DAMGO-evoked currents produced in oocytes expressing the Kir3.1(F137S)(N217D) mutant were smaller (387 ± 142 nA) than those produced by Kir3.1(F137S) (1170 ± 98 nA). We interpret the increased sensitivity of Kir3.1(F137S)(N217D) to endothelin and arachidonic acid as resulting from the introduction of the critical aspartate residue required for eicosanoid binding.
Although Kir 3.1 may form channels with endogenous Kir3.5, we did not exclude the less likely interpretation that the Kir3.1(F137S)(N217D) channel formed endothelin-sensitive heteromers with Kir3.5. We used a sequence alignment program to compare the Na+-insensitive Kir3.1 with the Xenopus homologue, Kir3.5. When Kir3.5 is aligned with Kir3.1, we note that Kir3.5 contains the critical aspartate that is required for Na+ activation of the channel (BCM Search Launcher, Baylor College of Medicine). Thus, we would expect that any heteromultimers composed of Kir3.1(F137S)(N217D) and Kir3.5 would be arachidonic acid-sensitive. This explains the sensitivity of Kir3.1 and Kir3.5 heteromultimers (Fig. 2). Future single channel recordings would resolve this question.
We explored the mechanism of endothelin receptor-induced inhibition of
mu opioid receptor activation of Kir3 in vitro. Although Xenopus oocytes are a complex expression system with
interacting signaling molecules, insights to Kir3 gating mechanisms are
evident from these results. The endothelin receptor is one member of
the Gq-coupled seven-transmembrane superfamily of receptors
(24). Other Gq receptors that regulate Kir3 by eicosanoid
production may also require specific gating components to exert effects
on the channel. For example, activation of the Gq-coupled
substance P receptors expressed in locus coeruleus neurons
inhibits Kir3 currents (26, 27). Although the mechanism of inhibition
was not established in that study, the results shown here may be
relevant. Eicosanoid regulation of Kir3 as described in this study may
be relevant to the pathological situations that occur during
inflammation. Because Kir3 has in important role in cardiac
excitability, cardiovascular disease may elicit an inflammatory
response to produce eicosanoids that inhibit Kir3 (28-32).
Nevertheless, the physiological significance of these findings needs to
be directly established.
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ACKNOWLEDGEMENTS |
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We thank Tracy Sherertz for assistance and Drs. Todd Scheuer and Tooraj Mirshahi for helpful discussion. We thank Dr. Lei Yu for the rat mu opioid receptor clone, Dr. Richard Kris for the human endothelin A receptor, Drs. Cesar Lebarca and Henry Lester for the Kir 3.1 and Kir 3.2 clones, Dr. John Adelman for the Kir 3.4 clone, and Dr. Diomedes Logothetis for the two Kir3 chimeras: Kir3.4(S143T) (1)/Kir3.1-(333-501) and Kir3.4(S143T)-(1-249)/Kir3.1-(244-501).
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FOOTNOTES |
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* This study was supported by United States Public Health Services Grant DA 04123 from the National Institute on Drug Abuse.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Pharmacology, Box 357280, University of Washington, Seattle, WA
98195-7280. Tel.: 206-543-4266; Fax: 206-685-3822; E-mail:
cchavkin@u.washington.edu.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M010097200
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ABBREVIATIONS |
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The abbreviations used are:
Kir3, G-protein-gated inwardly rectifying potassium channel;
PIP2, phosphatidylinositol 4,5-bisphosphate;
HETAR, human
endothelin A receptor;
MOR, mu opioid receptor;
DAMGO, D-Ala2Glyol, enkephalin;
Et-1, endothelin-1;
G2,
-2 subunit of the G-protein;
PLC, phospholipase C.
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