From the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
Received for publication, September 7, 2000
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ABSTRACT |
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The G protein-coupled inwardly rectifying
K+ channel, GIRK1/GIRK4, can be activated by
receptors coupled to the G Guanine nucleotide-binding protein coupled receptors
(GPCRs)1 play an important
role in many physiological processes, including the regulation of
excitability in cardiac and neuronal cells (1). The downstream
signaling pathways activated by GPCR agonists are dependent on the
class of heterotrimeric guanine nucleotide-binding (G) protein that is
coupled to the cytoplasmic domains of the receptor. For example, GPCRs
such as the dopamine 2 receptor (d2R) are coupled to the PTX-sensitive
Gi class of G proteins and thus mediate the inhibition of
adenylyl cyclase and a lowering of cellular cAMP levels. In contrast,
Gq-coupled receptors, such as the m1 muscarinic
acetylcholine receptor (mAChR), activate phospholipase C In many neurons, treatment with somatostatin enhances the conductance
of an inwardly rectifying K+ current, resulting in the
inhibition of neuron excitability (2, 3). This effect is mediated by a
PTX-sensitive G protein (3). In contrast, treatment with another
neurotransmitter, substance P, excites neurons by suppressing an
inwardly rectifying current through a PTX-insensitive G protein pathway
(4, 5). Velimirovic et al. (6) presented evidence that the
same inwardly rectifying channel is the recipient of these competing G
protein signals in locus coeruleus neurons, supporting the idea that
channels can be dually modulated by different classes of GPCRs.
Similar effects of GPCRs on ion channels have been documented in
defined expression systems using cloned channels and receptors. In
cardiac pacemaker cells, the m2 mAChR, which is coupled to the PTX
sensitive G The GIRK protein family contains five homologous members (GIRK1-5),
all of which contain two transmembrane domains flanking a hydrophobic
pore region, with both the N and C termini located in the cytoplasm
(7). GIRK channels function in the membrane as tetramers consisting of
two GIRK1 subunits and two subunits of one of the other GIRK family
members (GIRK2-5) (8-10). In cardiac cells, heteromultimeric
GIRK1/GIRK4 channels predominate (11).
The signaling pathway between the activating Gi-coupled
receptor and GIRK channels is membrane-delimited and dependent on the
direct binding of G Other groups have recently reported that Gq signaling can
inhibit GIRK channels (18-20). In this study, we corroborate and further their findings by investigating the effect of m1 mAChR stimulation on GIRK1/GIRK4 whole cell currents in a Xenopus
oocyte expression system. We found that stimulation of the
Gq-coupled m1 mAChR led to the suppression of GIRK1/GIRK4
currents and that this effect could be mimicked by a potent PKC
activator, PMA, and a Ca2+ ionophore. Analysis of chimeric
and mutant channels revealed that the GIRK4 subunit is sufficient for
responding to these Gq signals but not via direct
phosphorylation of a canonical PKC site on the channel itself.
Clones--
The following clones were used in these studies: rat
GIRK1, rat GIRK4, rat IRK2, human m1 mAChR, human d2R (long form), rat G Xenopus Oocyte Electrophysiology--
RNA synthesis and oocyte
preparation were performed as described previously (21). Defolliculated
oocytes were injected with 50 nl of a cRNA mixture containing the
appropriate combination of transcripts in these approximate amounts: 5 ng of GIRK1, 5 ng of GIRK4, 5 ng of d2R, 1 ng of m1 mAChR, and 2 ng of
IRK2. In Fig. 1B, basal GIRK currents were increased by the
injection of 2 ng of G
To minimize the contribution of endogenous currents, only oocytes with
>1µA of whole cell current at
Cell-attached patch recordings (see Fig. 1D) were performed
with 140 mM K+ saline (140 mM KCl,
10 mM HEPES, pH 7.4, 1 mM MgCl2) in
both the bath and the pipette. Quinpirole (100 µM) was
added to the pipette saline to activate GIRK channels. Glass pipettes
(Corning 8161) had tip resistances between 0.8 and 4 M Pharmacology--
To activate the d2R receptor, 10 µM (
Oocytes treated with calphostin C, the tyrosine kinase inhibitor
mixture (4 µM tyrphostin A-48, 4 µM
tyrphostin A-25, 1 µM lavendustin A, and 15 µM genistein), GF109203X, staurosporine, and H-89 were
soaked for 20-30 min prior to recording in 90 mM K+ recording saline containing 1:1000 dilutions of both
1000× drug (in Me2SO) and 30% pluronic F-127 (in
Me2SO). Pluronic F-127 alone had no effect on whole cell
GIRK1/GIRK4 currents (data not shown). PMA and Ca2+
ionophore were similarly dissolved and added to the recording solution
in the final concentrations given in the text.
4K+-1,2-bis(0-aminophenoxy)ethane- N,N,N',N'-tetraacetic
acid (BAPTA) was dissolved in 100 mM KCl to a final
concentration of 2 mM. A 50-nl bolus of this solution was
injected into oocytes 20-30 min before recording. Control oocytes were
similarly injected with 100 mM KCl. Similarly, KT5823 and
the inhibitory PKA peptide were dissolved in Ca2+-free OR-2
(96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, and 5 mM HEPES, pH 7.5) to final
concentrations of 20 µM and 200 nM,
respectively. A 50-nl bolus was injected 20-30 min before recording
and compared with oocytes injected with Ca2+-free OR-2 alone.
m1 mAChR Signaling Inhibits Both Basal and Activated GIRK1/4
Currents--
To test the effect of m1 mAChR stimulation on GIRK
currents, Xenopus oocytes expressing GIRK1, GIRK4, d2R, and
m1 mAChR were placed under two-electrode voltage clamp. GIRK currents
were first activated by the addition of 10 µM quinpirole.
Fifty seconds later, the m1 mAChR was stimulated by 100 nM
to 5 µM carbachol. Carbachol elicited a short lived
endogenous oocyte current that strongly inactivated at negative
potentials (Ref. 22 and data not shown). After this transient current
had subsided, we noted a carbachol-dependent inhibition in
GIRK current amplitude (Fig.
1A, left panel).
The right panel of Fig. 1A shows the average
percentage of suppression of activated GIRK currents over time
(n = 12). Interestingly, carbachol-treated GIRK
currents decreased at a rate that was three times faster than oocytes
that were not treated with carbachol. This effect was dependent on
signaling through the m1 mAChR because oocytes that were not injected
with cRNA encoding the m1 mAChR failed to exhibit a carbachol-induced
decrease in activated GIRK whole cell currents. (n = 3, data not shown). These results suggest that m1 mAChR, like the
metabotropic glutamate receptor 1a, bombesin 1, and endothelin 1 receptors (18-20), can inhibit GIRK activity.
To eliminate the possibility that the m1 mAChR acts by directly
inhibiting the d2R receptor itself, we next examined the effect of m1
mAChR stimulation on basal, nonreceptor-activated, GIRK1/GIRK4 currents. Oocytes injected with cRNA encoding GIRK1/4, m1 mAChR, G
The inwardly rectifying potassium channel, IRK2 (Kir2.2), is 45%
homologous to GIRK1 but does not respond to Gi-coupled
receptor signaling. We found that the large basal current
characteristic of IRK2 was also insensitive to Gq-coupled
receptor signaling; carbachol stimulation of the m1 mAChR had no effect
on IRK2 current amplitude (Fig. 1C). Similarly, the small
steady-state endogenous oocyte currents seen at
To determine whether second messenger molecules are necessary for m1
mAChR-mediated inhibition of GIRK1/4, we recorded GIRK currents in a
cell-attached patch configuration and tested for their sensitivity to
carbachol added to the bath. The left panel of Fig.
1D shows the openings of single GIRK channels in a
Xenopus oocyte patch. We found that carbachol added to the
bath inhibited channels located within the pipette, implying the
involvement of second messenger molecules (Fig. 1D). This is
in contrast to GIRK channel activation, which occurs by a
membrane-delimited pathway and thus requires addition of agonist to the
pipette; agonist addition to the bath does not activate GIRK currents
(12).
Overexpressed G Pharmacological Evidence Suggests a Role for PKC and
Ca2+ Signaling in m1 mAChR-mediated Suppression of
GIRK1/4--
Gq-coupled receptors, such as the m1 mAChR,
have been linked to many downstream effector pathways including
activation of phospholipase C, PKC, and tyrosine kinases (26-28). In
an effort to determine whether these pathways are involved in the
second messenger-dependent m1 mAChR-mediated suppression of
GIRK1/4 currents, oocytes injected with GIRK1, GIRK4, d2R, and m1 mAChR
mRNA were treated with various pharmacological agents.
PKC is a ubiquitous kinase known to be activated by
Gq-coupled receptors (29). Interestingly, 10 µM phorbol 12-myristate 13-acetate (PMA), a potent PKC
activator, mimicked the effect of m1 mAChR signaling on both activated
(Fig. 3A) and basal (Fig. 3B) GIRK1/4 currents. The kinetics of GIRK inhibition by PMA
were very similar to the receptor-mediated suppression (compare with Fig. 1). PMA treatment also inhibited IRK2 currents; however, this
inhibition took considerably longer to develop and occurred at a slower
rate than GIRK1/4 inhibition (Fig. 3C), suggesting that PMA
acts on these two channels by different mechanisms.
Treatment with a broad specificity serine/threonine kinase inhibitor,
staurosporine (3 µM), slowed both m1 mAChR-mediated and
agonist-independent channel suppression (Fig. 3D). H-89 (50 µM), another broad specificity kinase inhibitor, also
slowed m1 mAChR-mediated inhibition of activated currents (data not
shown). However, we were unable to further illustrate the involvement of PKC in GIRK1/4 inhibition through the use of specific PKC
inhibitors. The cell-permeable PKC inhibitor, calphostin C (up to 1 µM), did not affect m1 mAChR-mediated suppression of
GIRK1/4 (Fig. 3E). Another PKC inhibitor, GF109203X (1 µM), also failed to affect receptor-mediated inhibition
of GIRK currents. At higher concentrations (15 µM),
GF109203X blocked and disrupted GIRK1/4 currents (data not shown).
Because both H-89 and staurosporine inhibit a broad range of kinases
including PKC, PKA, and cGMP-dependent kinase at the concentrations used here, it is difficult to further define the role of
a specific kinase in GIRK suppression. Additional experiments utilizing
PKA and cGMP-dependent kinase inhibitors were unable to
identify a specific role for these kinases. Both a protein kinase A
inhibitory peptide (10 nM, Calbiochem) and KT5823 (1 µM, Calbiochem) failed to significantly affect m1
mAChR-mediated inhibition of GIRK channels (data not shown).
In addition to the activation of PKC and other kinases,
Gq-coupled receptors also strongly increase intracellular
Ca2+ levels in the cell. To explore the possibility that
Ca2+ signaling may mediate the Gq effect on
GIRK channels, we modulated intracellular Ca2+ levels in
oocytes with various pharmacological agents. Treatment with the
Ca2+ ionophore, A23187, inhibited GIRK1/4 currents in a
manner indistinguishable from m1 mAChR signaling or treatment with PMA (Fig. 3F). However, inhibition of Ca2+ signaling
by injection of the calcium chelating agent, BAPTA, did not
significantly affect m1 mAChR signaling to GIRK1/4, despite the
complete elimination of the transient Ca2+-activated
Cl
Tyrosine kinases have been implicated in the suppression of a
voltage-gated potassium channel by m1 mAChR signaling (28). However, we
have found no evidence for tyrosine kinase phosphorylation in the
regulation of GIRK channels by m1 mAChR. Treatment of oocytes with a
mixture of tyrosine kinase inhibitors did not affect the carbachol-induced inhibition of GIRK1/4 currents (data not shown).
GIRK4 May Be the Principle Target of These Inhibitory m1 mAChR
Signals--
Because pharmacology implicated a role for protein
kinases in m1 mAChR-mediated suppression of GIRK1/4 currents, we
searched for potential phosphorylation sites on the GIRK proteins that may mediate this effect. A ProSite data base search revealed 10 putative phosphorylation sites for both PKA and PKC in GIRK1 and three
PKA sites and six PKC sites in GIRK4 (30). Interestingly, the extended
C terminus of GIRK1 contains a particularly high density of
phosphorylation sites. To narrow our search, we were interested in
identifying the domains of the GIRK1 and GIRK4 proteins that mediate
the inhibitory effect of Gq signaling on GIRK currents. A
series of chimeric and mutant ion channels derived from GIRK1 and IRK2
were generated previously by Kunkel and Peralta (15) (Fig.
4A). We coexpressed these
chimeras with the m1 mAChR and tested whether they were responsive to
m1 mAChR signaling. The results from these experiments are summarized
in Fig. 4A. The d2R-activated current of both the GR2/GIRK4
and the
Interestingly, the chimeras that require coinjection of cRNA encoding
the GIRK4 subunit to produce visible whole cell currents were
suppressed upon m1 mAChR stimulation. This correlation suggested that
the GIRK4 subunit itself may be responsive to Gq signals. To test this hypothesis, we took advantage of a mutation in the pore
region of GIRK4, a serine to phenylalanine substitution at amino acid
143, that allows wild-type and S143F mutant GIRK4 subunits to form
functional homomeric channels (31, 32). We found that GIRK4/GIRK4(S143F) homomeric channels are indeed inhibited by stimulation of the m1 mAChR (Fig. 4B) in a similar manner to
GIRK1/4 heteromeric channels. Thus, the GIRK4 subunit is sufficient to respond to Gq signals.
Phosphorylation of a Canonical PKC Site on the Channel Is Not
Necessary for GIRK Current Inhibition by m1 mAChR--
Our
pharmacological analysis implicates serine/threonine kinases in the
regulation of GIRK channels by m1 mAChR signaling. Because GIRK
channels contain numerous putative PKA and PKC phosphorylation sites,
we wondered whether phosphorylation of GIRK1 or GIRK4 is required for
GIRK current inhibition by m1 mAChR signaling. To address this
question, we generated site-specific mutations to eliminate potential
phosphorylation sites and tested these mutants for their ability to be
suppressed by m1 mAChR. In particular, we took advantage of the
GIRK4(S143F) pore mutation that allows GIRK4 to form homomeric channels
and used site-directed mutagenesis to eliminate the canonical PKC
phosphorylation sites found in this protein. GIRK4 contains six
putative PKC sites ((S/T)X(R/K)), five of which are
predicted to be accessible to intracellular kinases. The sixth PKC site
is located immediately adjacent to the pore helix (H5 region),
N-terminal to the second transmembrane domain (Fig.
5A). GIRK4 homomeric channels
in which all five intracellular PKC sites have been mutated to alanine,
designated GIRK4( In this paper, we have shown that a cloned inward rectifier,
GIRK1/GIRK4, is functionally coupled to the m1 mAChR in a
Xenopus oocyte expression system. Thus, the regulation of
GIRK1/4 conductance is mediated in opposing manners by different
classes of GPCRs; it responds to an activation signal from
Gi-coupled receptors such as d2R and an inhibitory signal
from the Gq-coupled m1 mACh receptor. Other studies have
demonstrated that stimulation of the m1 mAChR increases excitability in
some cells by suppressing a voltage-gated K+ channel, Kv1.2
(28). Our findings suggest that GIRK1/4 may also contribute to these m1
mAChR-mediated stimulatory signals by lowering the membrane
permeability to K+, thus allowing the cell to reach firing
threshold more quickly.
It is interesting to speculate that the inhibition of GIRK currents by
Gq-coupled receptors may be partially responsible for determining the specificity of GIRK activation. Although all G proteins
release G Other groups have also shown that GIRK channels can be suppressed by
Gq-coupled receptors, including the metabotropic
glutamate receptor 1a, the endothelin A receptor, and the bombesin 1 receptor (18-20). Thus, it is likely that all Gq-coupled
receptors are capable of inhibiting GIRK currents, just as GIRK
currents are activated by any Gi-coupled receptor. However,
the mechanism of Gq-mediated suppression of GIRK remains unclear.
Activating protein kinase C (via PMA) or increasing cytosolic
Ca2+ levels with A23187 mimicked the suppression of GIRK
currents elicited by stimulation of the m1 mAChR with carbachol.
Surprisingly, in our experiments, specific PKC targeting drugs that
inactivate typical PKC subtypes failed to prevent or slow m1
mAChR-mediated suppression of GIRK1/4. However, broad specificity
kinase inhibitors slowed both m1 mAChR-mediated and agonist-independent
inhibition of GIRK1/4 currents, suggesting that serine/threonine
kinases are likely to be involved in GIRK inhibition by
Gq-coupled receptors. There are many different classes of
PKC enzymes, all of which prefer similar phosphorylation site sequences
(33). It is possible that an atypical PKC family member is responsible
for mediating the m1 mAChR effect on GIRK channels. In fact, Sharon
et al. (18) suggested PKC-µ, an atypical PKC, as a likely
candidate. Stevens et al. (19) also found pharmacological
evidence for the involvement of PKC. These pharmacological data led
many of these groups to speculate that PKC may directly phosphorylate
the channel itself resulting in a subsequent current inhibition.
This mechanism is known to inhibit other channels, including an
IRK family member, Kir2.3 (34). However, our results suggest that this
mechanism is unlikely to explain Gq-mediated inhibition of
GIRK currents because a mutant homomeric GIRK4 channel that does not
contain accessible canonical PKC sites continues to respond to
Gq signaling.
Because stimulation of m1 mAChR can inhibit both basal and
receptor-activated GIRK currents, m1 mAChR appears to regulate GIRK
channel activity at a level downstream of the activating dopamine 2 receptor. GIRK activation occurs by a membrane-delimited pathway
involving direct Gi subunit. An opposing
role for G
q receptor signaling in GIRK regulation has
only recently begun to be established. We have studied the effects of
m1 muscarinic acetylcholine receptor (mAChR) stimulation, which is
known to mobilize calcium and activate protein kinase C (PKC) by a
G
q-dependent mechanism, on whole cell
GIRK1/4 currents in Xenopus oocytes. We found that
stimulation of the m1 mAChR suppresses both basal and dopamine 2 receptor-activated GIRK 1/4 currents. Overexpression of G
subunits attenuates this effect, suggesting that increased binding of
G
to the GIRK channel can effectively compete with the
Gq-mediated inhibitory signal. This Gq signal
requires the use of second messenger molecules; pharmacology implicates
a role for PKC and Ca2+ responses as m1 mAChR-mediated
inhibition of GIRK channels is mimicked by PMA and Ca2+
ionophore A23187. We have analyzed a series of mutant and chimeric
channels suggesting that the GIRK4 subunit is capable of responding to
Gq signals and that the resulting current inhibition does
not occur via phosphorylation of a canonical PKC site on the channel itself.
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ABSTRACT
INTRODUCTION
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DISCUSSION
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, resulting
in the activation of protein kinase C (PKC) and an increase in
cytoplasmic Ca2+ levels.
i protein, is associated with the activation of the G protein-coupled inwardly rectifying K+ channel
(GIRK). GIRK channels are characterized by a low basal level of
activity that is dramatically increased upon stimulation of
Gi-coupled receptors. The activation of these channels
results in a decrease in cellular excitability by hyperpolarizing the cell, thus lengthening the time between action potentials.
subunits to the cytoplasmic domains of both
GIRK1 and GIRK4 (12-16). Recently, it has been suggested that G
binding to GIRK channels may stimulate channel activity by increasing
the affinity of the cytoplasmic regions of GIRK for
phosphatidylinositol 4,5-bisphosphate (17), an association that appears
to be required for the activity of all inwardly rectifying potassium
channels (17).
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ABSTRACT
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DISCUSSION
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i2, murine G
1, and murine
G
2. Chimeric GIRK1/IRK2 channels were generated as
described previously (15). Mutant channels were generated using the
QuikChange site-directed mutagenesis kit (Stratagene) protocol.
1 and 1 ng of G
2. A
larger amount of G
RNA was injected for Fig. 2 (~8 ng of
G
1, 3 ng of G
2). Oocytes were recorded
3-5 days later by two-electrode voltage clamp in 90 mM K+ saline (90 mM KCl, 3 mM
MgCl2, 5 mM HEPES, pH 7.4) as described previously (15). Currents from a minimum of five oocytes were recorded
for each treatment.
80 mV were used. Endogenous oocyte
current amplitudes at
80 mV are generally less than 0.2 µA. Oocytes
were held at 0 mV and pulsed to
80 mV for 500 ms at 10-s intervals.
Currents were also monitored at 0 and +40 mV. Data from multiple
oocytes were normalized by computing the percent suppression: % suppression =
((X
A)/A) × 100, where X
is the current amplitude at a given time point and A is the
current amplitude immediately before carbachol addition and after d2R
activation (if applicable).
.
Cell-attached patches containing multiple channels were held at 0mV.
Every 5 s, the voltage was pulsed to
60 mV for 2700 ms, followed
by a 460-ms pulse to +60 mV. Carbachol (1 µM) was added
to the bath after 100 s and allowed to mix by diffusion. Data were
analyzed using Fetchan (Axon Instruments) by calculating the percent
total channel activity in a 100-s window beginning 100 s after
carbachol (or control saline) addition relative to an identical window
before carbachol (or saline) addition.
)-quinpirole hydrochloride (Research Biochemicals
International) was added to the recording solution. Similarly, the m1
mAChR was activated by addition of 100 nM to 10 µM carbachol. The concentration of carbachol used in each
oocyte batch was chosen by normalizing the endogenous Ca2+-activated chloride transient current evoked by m1
mAChR stimulation in oocytes (22). A concentration of carbachol that
evoked a visible but short lived (<30 s) transient current was used.
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ABSTRACT
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Fig. 1.
The m1 mAChR mediates a second
messenger-dependent inhibition of both activated and basal
GIRK1/GIRK4 but not IRK2 currents. Oocytes were held at 0 mV and
pulsed to 80 mV at 10-s intervals. The left panels show
the current amplitude over time from one representative oocyte
subjected to control saline (open squares) or carbachol
treatment (filled diamonds). Addition of quinpirole and
carbachol to the recording solution is represented by the
filled and open-headed arrows, respectively. The
right panels show the percentage of suppression of current
amplitude over time for a population of oocytes. At time 0, control
saline (open squares) or carbachol (filled
diamonds) was added. Both quinpirole-activated (A) and
basal (B) GIRK1/GIRK4 currents are inhibited by m1 mAChR
stimulation. In contrast, IRK2, an inwardly rectifying potassium
channel that is not responsive to Gi-coupled receptors, is
unaffected by m1 mAChR stimulation (C). GIRK1/4 channel
activity at
60 mV was recorded in patch configuration from a
Xenopus oocyte (D, left panel).
Downward deflections reflect the opening of a GIRK channel. At the end
of each trace, the voltage was stepped to +60 mV to ensure the channel
was inwardly rectifying. GIRK channel activity significantly decreased
after addition of carbachol to the bath (and subsequent stimulation of
m1 mAChR signaling), implying a role for second messenger molecules.
This effect was quantified by calculating the percentage of suppression
of total channel current during a 100-s window after addition of saline
(no agonist) or carbachol relative to an identical window before saline
or carbachol addition (D, right panel).
1, and G
2 were screened for a high level
of basal GIRK current. As previously reported by Kovoor et
al. (23), we found that the basal current amplitude of GIRK1/GIRK4
decreased slowly in the absence of any agonists after placing the
oocytes into high potassium saline. However, when carbachol (0.5-1.0
µM) was added to the bath, the basal GIRK current
amplitude was significantly inhibited (Fig. 1B) with similar
kinetics to those seen in the inhibition of d2R-activated GIRK
currents. This finding suggests that m1 mAChR-mediated inhibition of
GIRK1/4 occurs downstream of the activating receptor.
80 mV were also
insensitive to m1 mAChR signaling (n = 5; data not shown).
Can Compete with the Inhibitory
Gq Signal--
Many groups have reported that
overexpression of G
subunits results in an increase in basal
GIRK1/4 current, presumably because of increased binding of G
to
the cytoplasmic regions of the channel and subsequent channel
activation (8, 24, 25). However, heterotrimeric G proteins (G
)
do not activate GIRK. The previous experiments described here utilized
only G proteins that are endogenous to the oocyte (although small
amounts of G
1 and G
2 cRNA were injected
in Fig. 1B). Interestingly, we found that GIRK currents in
oocytes that were injected with G
1 and G
2
cRNA were less responsive to Gq signaling than oocytes that
also expressed the G
i2 subunits, suggesting that free
G
subunits are capable of reversing or preventing Gq
inhibition (Fig. 2, A and
B). Notably, the slower agonist-independent GIRK suppression
was similarly affected by the overexpression of G protein subunits
(Fig. 2B). Basal GIRK1/4 current was significantly decreased
by the overexpression of G
i2, suggesting that
G
i2 protein was expressed and functional (Fig.
2D). Furthermore, the amplitude of the transient
Ca2+ activated Cl
current was comparable in
the oocytes expressing G
and G
i2
, indicating
similar levels of m1 mAChR stimulation under these two conditions (Fig.
2C). These experiments show that high levels of free G
subunits have an inhibitory effect on Gq receptor signaling
to GIRK1/4.
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Fig. 2.
Overexpressed
G attenuates m1 mAChR-mediated
inhibition of GIRK1/4. Xenopus oocytes were injected
with GIRK1/4 and m1 mAChR cRNA and then injected a second time with
dopamine receptor and either G
1
2 RNA
alone or G
i2 and G
1
2 cRNA
(A). The percentage of suppression over time as the result
of carbachol addition for each of these batches of oocytes was
determined (B). Overexpression of
G
1
2 subunits dramatically inhibits
suppression of basal GIRK1/4 currents upon stimulation of the m1 mAChR
by carbachol (B, open circles). This effect is
rescued by the additional expression of G
i2
(B, closed circles). C, the maximal
Ca2+-activated Cl
current at 0 mV elicited by
m1 mAChR stimulation in each batch of oocytes. D, basal
current amplitude at
80 mV of GIRK1/4 currents in each batch of
oocytes.
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Fig. 3.
Serine/threonine kinases and Ca2+
signaling are involved in GIRK1/4 inhibition. A-C,
d2R-activated GIRK1/4 currents (A), basal GIRK1/4 currents
(B), or IRK2 currents (C) at 80 mV were
recorded from oocytes that had been treated with 10 µM
PMA (closed diamonds) or received no treatment (open
squares) at time 0. The percentage of suppression was determined
by comparing currents to the current recorded immediately before PMA
treatment. Activated channels were treated with quinpirole 50 s
before PMA addition. D, pretreatment with a broad
specificity kinase inhibitor, staurosporine (3 µM), slows
both agonist independent (squares) and carbachol-mediated
(diamonds) inhibition of d2R-activated GIRK1/GIRK4.
E, calphostin C (1 µM), a specific PKC
inhibitor, does not affect inhibition of activated GIRK1/4 currents by
m1 mAChR stimulation. F and G, calcium signaling
inhibits GIRK1/4 currents but is not required for m1 mAChR-mediated
channel inhibition. Whole cell currents at
80 mV were recorded from
oocytes injected with RNA encoding GIRK1, GIRK 4, d2R, and m1 mAChR.
Calcium ionophore A23187 mimicked the effect of m1 mAChR stimulation on
d2R-activated GIRK1/4 currents (F). In contrast,
microinjection of the calcium chelating agent, BAPTA,
effectively eliminated the Ca2+-activated Cl
current but did not significantly affect carbachol-mediated suppression
of GIRK1/GIRK4 (G).
current (Fig. 3G).
N1/GIRK4 mutant channels were suppressed upon stimulation of
the m1 mAChR with carbachol. Taken together, these results suggest that
the serine/threonine-rich distal N-terminal (amino acids 1-24) and C-terminal (amino acids 357-501) regions of GIRK1 are not required for
GIRK inhibition by m1 mAChR. Thus, it appears that the numerous potential phosphorylation sites in these regions are not necessary for
channel responsiveness to Gq signaling. In contrast, the
large basal (nonreceptor activated) currents of the GR3 and GR7.1
chimeras were insensitive to Gq signaling, suggesting that
amino acids 1-84 and 290-501 of GIRK1 are not capable of conferring
Gq sensitivity onto IRK2.
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Fig. 4.
Identification of channel domains responsive
to Gq signaling. A, chimeric channels
between GIRK1 and IRK2 (15) were used to determine the channel regions
necessary for m1 mAChR-mediated suppression. The channel protein
contains three major domains: the N terminus (N), the pore
region (including the two transmembrane domains) (TM1,
H5, and TM2), and the C terminus (consisting of
C1 and C2). Chimeras that require coinjection of
GIRK4 to produce visible whole cell currents are designated with a + in
the left columns. These same chimeras were also sensitive to
m1 mAChR signaling (right column). Currents from GIRK1, GR2,
and N1 channels were activated with quinpirole prior to addition of
carbachol. IRK2, GR7.1, and GR3 displayed large basal currents and were
not treated with quinpirole. B, GIRK4(S143F) mutant proteins
can form functional channels with wild-type GIRK4. These homomeric
GIRK4 channels are suppressed upon stimulation of the m1 mAChR by
carbachol. Oocytes were injected with RNA encoding GIRK4, GIRK4(S143F),
d2R, and m1 mAChR. Currents were activated with 10 µM
quinpirole 50 s before addition of control saline (open
triangles) or carbachol (filled circles) at time
0.
PKC), did not produce current in oocytes coinjected
with the dopamine 2 receptor. However, the subsequent injection of
G
1 and G
2 cRNAs produced whole cell GIRK
currents of greater than 1 µA from these mutant channels.
Interestingly, GIRK4(
PKC) channels are inhibited by Gq
stimulation (Fig. 5B) to a similar extent as wild-type GIRK4 homomeric channels. This result suggests that direct PKC
phosphorylation of the channel is not necessary for
Gq-mediated inhibition.
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Fig. 5.
Phosphorylation of a canonical PKC site on
the GIRK channel is not required for m1 mAChR-mediated inhibition.
A, the GIRK4( PKC) mutant construct. Putative PKC sites
are depicted by black boxes. The five starred PKC
phosphorylation sites were mutated to alanine. The remaining PKC site,
located between the hydrophobic pore region and the second
transmembrane domain, is not likely to be accessible to intracellular
proteins. B, GIRK4(
PKC) is susceptible to inhibition by
Gq signaling. GIRK4(
PKC) and GIRK4(
PKC, S143F) were
coexpressed in Xenopus oocytes along with d2R, m1 mAChR,
G
1, and G
2. The resulting homomeric
GIRK4(
PKC) channels (open circles) were indistinguishable
from wild-type GIRK4 homomeric channels (filled circles) in
their ability to be inhibited by m1 mAChR stimulation. However, the
smaller whole cell current produced by the GIRK4(
PKC) channels
caused the Ca2+-activated Cl
transient
current to be a larger percentage of the mutant channel current than
the wild-type GIRK4 current.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits upon activation, only Gi-coupled receptors are capable of activating GIRK. Interestingly, GIRK channels
do not seem to prefer particular combinations of
and
subunits,
suggesting that only the identity of the G
subunit is important for
determining specificity (14). Gi-coupled receptors often
have inhibitory physiological effects and are not generally thought to
activate many cellular kinases. In contrast, other G proteins such as
Gq and Gs stimulate many kinases, including PKC
and PKA. Perhaps activation of these kinases inhibits GIRK channels by
lowering their affinity for G
, thus protecting them from
subsequent activation.
binding to the channel (12, 13, 15, 16, 35).
Thus, it seems likely that m1 mAChR signaling targets either the
activating heterotrimeric G protein or the GIRK channel itself. The
exact nature of basal GIRK activity remains unclear, and it is a source
of argument in the field whether GIRK channels display G
protein-independent activity (see Ref. 7). In oocytes, the basal
current does not depend on the amount of expressed receptor, and
~80% of the basal GIRK current is sensitive to PTX toxin, which
targets the Gi heterotrimeric G protein (8, 36, 37).
Additionally, coexpression of
ARK-PH domain or other G
binding
molecules can reduce basal GIRK activity (14, 24, 38, 39). These
findings suggest that a significant fraction of the basal GIRK current
results from basal G protein activity. Thus, it remains possible that
Gq signaling inhibits GIRK currents by inactivating the
Gi protein rather than directly targeting the channel itself.
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ACKNOWLEDGEMENTS |
---|
I thank Maya Kunkel for providing the
GIRK1/IRK2 chimera clones and assistance in the early phases of this
project. I also thank Dr. N. Gautam for generously providing the
G1 and G
2 cDNA clones, the scientist
who provided the d2R clone, and Guido Guidotti and Joel Bard for
valuable suggestions about this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Department of Molecular and Cellular Biology at Harvard University.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.
Supported by a predoctoral training grant from the National
Institutes of Health. To whom correspondence should be addressed. Present address: Genetics Institute, 87 Cambridge Park Dr., Cambridge, MA 02140. E-mail: jjhill@post.harvard.edu.
Ernest G. Peralta died on May 17, 1999. He is remembered as an
extraordinary scientist, mentor, and colleague.
Published, JBC Papers in Press, November 11, 2000, DOI 10.1074/jbc.M008213200
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ABBREVIATIONS |
---|
The abbreviations used are: BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; GPCR, G protein-coupled receptor; GIRK, G protein-coupled inwardly rectifying K+ channel; PKC, protein kinase C; mAChR, muscarinic acetylcholine receptor; PMA, phorbol 12-myristate 13-acetate; d2R, dopamine 2 receptor; PTX, pertussis toxin; PKA, cAMP-dependent kinase.
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REFERENCES |
---|
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---|
1. | Iismaa, T. P., and Shine, J. (1992) Curr. Opin. Cell. Biol. 4, 195-202[Medline] [Order article via Infotrieve] |
2. | Mihara, S., North, R. A., and Surprenant, A. (1987) J. Physiol. (Lond.) 390, 335-355[Abstract] |
3. | Inoue, M., Nakajima, S., and Nakajima, Y. (1988) J. Physiol. (Lond.) 407, 177-198[Abstract] |
4. | Stanfield, P. R., Nakajima, Y., and Yamaguchi, K. (1985) Nature 315, 498-501[Medline] [Order article via Infotrieve] |
5. | Nakajima, Y., Nakajima, S., and Inoue, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3643-3647[Abstract] |
6. | Velimirovic, B. M., Koyano, K., Nakajima, S., and Nakajima, Y. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1590-1594[Abstract] |
7. | Dascal, N. (1997) Cell Signal 9, 551-573[CrossRef][Medline] [Order article via Infotrieve] |
8. | Chan, K. W., Langan, M. N., Sui, J. L., Kozak, J. A., Pabon, A., Ladias, J. A., and Logothetis, D. E. (1996) J. Gen. Physiol. 107, 381-397[Abstract] |
9. | Kofuji, P., Davidson, N., and Lester, H. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6542-6546[Abstract] |
10. |
Silverman, S. K.,
Lester, H. A.,
and Dougherty, D. A.
(1996)
J. Biol. Chem.
271,
30524-30528 |
11. | Krapivinsky, G., Gordon, E. A., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141[CrossRef][Medline] [Order article via Infotrieve] |
12. | Soejima, M., and Noma, A. (1984) Pflugers Arch. Eur. J. Physiol. 400, 424-431[Medline] [Order article via Infotrieve] |
13. | Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E. J., and Clapham, D. E. (1987) Nature 325, 321-326[CrossRef][Medline] [Order article via Infotrieve] |
14. | Wickman, K. D., Iniguez-Lluhi, J. A., Davenport, P. A., Taussig, R., Krapivinsky, G. B., Linder, M. E., Gilman, A. G., and Clapham, D. E. (1994) Nature 368, 255-257[CrossRef][Medline] [Order article via Infotrieve] |
15. | Kunkel, M. T., and Peralta, E. G. (1995) Cell 83, 443-449[Medline] [Order article via Infotrieve] |
16. |
Krapivinsky, G.,
Krapivinsky, L.,
Wickman, K.,
and Clapham, D. E.
(1995)
J. Biol. Chem.
270,
29059-29062 |
17. | Huang, C. L., Feng, S., and Hilgemann, D. W. (1998) Nature 391, 803-806[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Sharon, D.,
Vorobiov, D.,
and Dascal, N.
(1997)
J. Gen. Physiol.
109,
477-490 |
19. |
Stevens, E. B.,
Shah, B. S.,
Pinnock, R. D.,
and Lee, K.
(1999)
Mol. Pharmacol.
55,
1020-1027 |
20. | Rogalski, S. L., Cyr, C., and Chavkin, C. (1999) J. Neurochem. 72, 1409-1416[CrossRef][Medline] [Order article via Infotrieve] |
21. | Kunkel, M. T., and Peralta, E. G. (1993) EMBO J. 12, 3809-3815[Abstract] |
22. | Dascal, N., Landau, E. M., and Lass, Y. (1984) J. Physiol. (Lond.) 352, 551-574[Abstract] |
23. |
Kovoor, A.,
Henry, D. J.,
and Chavkin, C.
(1995)
J. Biol. Chem.
270,
589-595 |
24. | Reuveny, E., Slesinger, P. A., Inglese, J., Morales, J. M., Iniguez-Lluhi, J. A., Lefkowitz, R. J., Bourne, H. R., Jan, Y. N., and Jan, L. Y. (1994) Nature 370, 143-146[CrossRef][Medline] [Order article via Infotrieve] |
25. | Velimirovic, B. M., Gordon, E. A., Lim, N. F., Navarro, B., and Clapham, D. E. (1996) FEBS Lett. 379, 31-37[CrossRef][Medline] [Order article via Infotrieve] |
26. | Peralta, E. G., Ashkenazi, A., Winslow, J. W., Ramachandran, J., and Capon, D. J. (1988) Nature 334, 434-437[CrossRef][Medline] [Order article via Infotrieve] |
27. | Ashkenazi, A., Peralta, E. G., Winslow, J. W., Ramachandran, J., and Capon, D. J. (1989) Trends Pharmacol. Sci. 10 (suppl.), 16-22 |
28. | Huang, X. Y., Morielli, A. D., and Peralta, E. G. (1993) Cell 75, 1145-1156[Medline] [Order article via Infotrieve] |
29. | Newton, A. C. (1997) Curr. Opin. Cell. Biol. 9, 161-167[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Hofmann, K.,
Bucher, P.,
Falquet, L.,
and Bairoch, A.
(1999)
Nucleic Acids Res.
27,
215-219 |
31. |
Chan, K. W.,
Sui, J. L.,
Vivaudou, M.,
and Logothetis, D. E.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14193-14198 |
32. |
Vivaudou, M.,
Chan, K. W.,
Sui, J. L.,
Jan, L. Y.,
Reuveny, E.,
and Logothetis, D. E.
(1997)
J. Biol. Chem.
272,
31553-31560 |
33. |
Nishikawa, K.,
Toker, A.,
Johannes, F. J.,
Songyang, Z.,
and Cantley, L. C.
(1997)
J. Biol. Chem.
272,
952-960 |
34. |
Zhu, G.,
Qu, Z.,
Cui, N.,
and Jiang, C.
(1999)
J. Biol. Chem.
274,
11643-11646 |
35. | Huang, C. L., Slesinger, P. A., Casey, P. J., Jan, Y. N., and Jan, L. Y. (1995) Neuron 15, 1133-1143[Medline] [Order article via Infotrieve] |
36. | Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1993) Nature 364, 802-806[CrossRef][Medline] [Order article via Infotrieve] |
37. | Dascal, N., Lim, N. F., Schreibmayer, W., Wang, W., Davidson, N., and Lester, H. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6596-6600[Abstract] |
38. |
Nair, L. A.,
Inglese, J.,
Stoffel, R.,
Koch, W. J.,
Lefkowitz, R. J.,
Kwatra, M. M.,
and Grant, A. O.
(1995)
Circ. Res.
76,
832-838 |
39. | Yamada, M., Ho, Y. K., Lee, R. H., Kontanill, K., Takahashill, K., Katadall, T., and Kurachi, Y. (1994) Biochem. Biophys. Res. Commun. 200, 1484-1490[CrossRef][Medline] [Order article via Infotrieve] |