(Received for publication, December 19, 1995)
From the
The G-protein-gated inward-rectifying K channel
GIRK1 has been demonstrated in heart and brain. These tissues also both
express the M
, M
, and M
muscarinic
acetylcholine receptors (mAChR) (Gadbut, A. P., and Galper, J. B.(1994) J. Biol. Chem. 269, 25823-25829). Only the M
mAChR has been demonstrated to couple to GIRK1 (Kubo, Y.,
Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y.(1993) Nature 264, 802-806). In this study we determined the
specificity of coupling of the M
and M
mAChR to
a new GIRK1 cloned from a chick brain cDNA library. This clone codes
for a 492-amino acid protein that is 93% identical to rat GIRK1 and is
expressed in brain, atrium, and ventricle, but not skeletal muscle. In Xenopus laevis oocytes co-expression of GIRK1 with either the
chick M
or M
mAChR gave carbamylcholine (10
µM)-stimulated K
currents of 308 ±
26 nA and 298 ± 29 nA, respectively, which were both
Ba
- and pertussis toxin-sensitive. Activation of the
M
receptor produced 2382 ± 478 nA of current which
was insensitive to Ba
and pertussis toxin, but was
85% inhibitable by the Cl
channel blocker
5-nitro-2-(3-phenylpropylamino)benzoic acid (10-20
µM) consistent with coupling to an endogeneous
Ca
-activated Cl
channel via a
phosphatidylinositol-dependent mechanism. Co-expression of the cardiac
inward rectifier CIR with chick M
or M
mAChR
and GIRK1 increased currents more than 10-fold, but had no effect on
specificity of coupling. These data demonstrate a new function for the
M
mAChR and a high degree of specificity for coupling of
each receptor subtype to GIRK1.
Muscarinic acetylcholine receptors (mAChR) ()have
been demonstrated to play an important role in both the heart and the
central nervous system. The M
isoform of the mAChR has been
shown to be coupled to an inward-rectifying K
channel
in the atrium that is responsible for mediating the negative
chronotropic response of the heart (for review, see (1) and (2) ). The M
mAChR isoform is expressed at high
levels in the basal ganglia(3, 4) , and more recently
M
mRNA has been demonstrated in the
heart(5, 6) . The physiological role of M
receptors in these tissues is not well understood. M
and M
isoforms of the muscarinic receptor expressed
in Chinese hamster ovarian cells were both coupled by a pertussis
toxin-sensitive G-protein to inhibition of adenylate cyclase activity
and to a modest stimulation of phospholipase C activity. M
and M
isoforms of the muscarinic receptor expressed
in Chinese hamster ovarian (CHO) cells were coupled via a pertussis
toxin-insensitive G-protein, such as G
, to a robust
stimulation of inositol trisphosphate production and were not coupled
to inhibition of adenylate cyclase(7) . Whether the similarity
in M
and M
coupling extends to stimulation of
the inward-rectifying acetylcholine-stimulated K
channel has not been determined.
Studies of inside-out patches
from membranes of cultured chick and rat atrial cells have demonstrated
that GTP-dependent release of from heterotrimeric G-proteins
activates inward-rectifying K
channels(8) .
Recently the G-protein-coupled inward-rectifying K
channel (GIRK1) has been cloned from rat and shown to be
activated by acetylcholine in Xenopus laevis oocytes
co-expressing the M
muscarinic receptor,
,
, and
(9, 10) . Activation of this channel
by acetylcholine has been mimicked by adding various combinations of
different
and
subunits to inside-out patches of rat atrial
myocytes(11) .
Recently Krapivinsky et al.(12) have demonstrated that antibodies to GIRK1
co-immunoprecipitated two proteins from atrial myocytes. One of the
co-immunoprecipitating proteins was GIRK1, and the second protein, when
purified and cloned, was determined to be a cardiac inward rectifier
(CIR), which was strikingly similar to the rcK-1 reported
by Ashford et al.(13) . Co-expression of CIR and GIRK1
in Xenopus oocytes resulted in an enhanced inward rectifier
channel with properties more closely resembling the atrial
acetylcholine-sensitive K
channel. In the present
study various muscarinic receptor isoforms are co-expressed in frog
oocytes with a new GIRK1 from chick brain that is present in atrium and
ventricle. Experiments demonstrate that both M
and M
mAChR are coupled via a similar mechanism to activation of GIRK1.
The data suggest a high degree of specificity of a given mAChR isoform
for activation of GIRK1 and the lack of an effect of co-expression of
CIR on the specificity of coupling.
Figure 1: Restriction endonuclease map and amino acid comparison of chick GIRK1. A, restriction map of the 2.5-kilobase pair cDNA coding for the chick GIRK1. Crossbars represent the coding region. B, amino acid comparison between the chick and rat GIRK1. The asterisk indicates amino acids that are identical to the chick GIRK1, and dots signify gaps introduced to align sequences. The predicted transmembrane domains and the H5 region are indicated by rectangular boxes.
Figure 2: RNase protection analysis of the expression of chick GIRK1 in chick tissues. Total RNA from the brain, atrium, ventricle, and skeletal muscle of chick embryos 17 days in ovo were subjected to RNase protection analysis to yield a 307-base pair product. The full-length riboprobe undigested with ribonuclease is shown in the left-hand lane. The probe contained an additional 72 nucleotides of Bluescript II on the 5` end of the GIRK1 transcript, which was not protected. DNA sequencing ladders run as standards in each experiment (not shown) were used to determine fragment sizes indicated on the left of the panel. Antisense riboprobes were hybridized to 15 µg of total RNA or tRNA as control. Autoradiograms represent exposures of 24 h.
Figure 3:
Functional characterization of the
coupling of different mAChR subtypes to GIRK1 ± CIR in X.
laevis oocytes by two-electrode voltage clamp. Inward currents
were evoked by 10 µM carbamylcholine in a 96 mM K-containing solution. A, B, D, E, G, H,
and J represent individual oocytes injected with cRNAs coding
for M
mAChR and GIRK1 (A); M
mAChR,
GIRK1, and CIR (B); M
mAChR and GIRK1 (D); M
mAChR, GIRK1, and CIR (E); M
mAChR and GIRK1 (G); M
mAChR, GIRK1, and CIR (H); M
and GIRK1 (J). C, F, and I show IV relationships for carbamylcholine-stimulated
currents in oocytes co-injected with M
, mAChR, GIRK1
± CIR (C); M
, GIRK
± CIR (F); M
mAChR, GIRK1 ± CIR (I);
GIRK1 alone (
); GIRK1 and CIR (
).
indicates the
addition of carbamylcholine (10 µM);
indicates the
addition of 1.0 mM Ba
. As shown in E, we occasionally observed a small
Ba
-sensitive current in oocytes injected with
M
, GIRK1, and CIR cRNA. This Ba
-sensitive
current was observed with or without co-expression of
CIR.
Although
M mAChR have been shown to activate different second
messenger systems than M
and M
mAChR (7) , stimulation of M
receptors does result in
release of
subunits from heterotrimeric G-proteins, which
could stimulate GIRK1 in the absence of the M
receptor.
Furthermore, since chick GIRK1 has a significantly different amino
terminus than rat, this could effect the specificity of coupling to
muscarinic receptors. Phosphatidylinositol-coupled receptors like
M
have been shown to produce agonist-dependent stimulation
of Ca
-activated Cl
channels in Xenopus oocytes(23) . We used this system to assess
whether the M
mAChR might couple to the chick GIRK1 isoform
and/or activate the endogeneous Cl
channel. In
oocytes expressing only the M
mAChR, carbamylcholine (10.0
µM) stimulated large rapidly activating and inactivating
inward currents of 3417 ± 1500 nA. In oocytes expressing both
M
and GIRK1 carbamylcholine stimulated a similar large
rapidly activating and inactivating inward current of 2382 ± 478
nA (n = 18) (Fig. 3D and Table 1), which was not significantly different from that with
M
alone. Both these currents were Ba
- and
pertussis toxin-insensitive. This distinctive current profile, the
sensitivity of the current to inhibition by NPPB, a Cl
channel blocker (from 2,382 ± 478 nA to 364 ± 100
nA, n = 5, p < 0.05), together with the
nonrectifying nature of the IV curve (Fig. 3F),
provided strong evidence that M
AChR coupled only to the
endogeneous Cl
channel rather than a K
channel(23) . To determine whether the lack of
carbamylcholine stimulation of GIRK1 in oocytes expressing M
was due to the inability of endogenous G-proteins to couple
M
to GIRK1, we assessed the effect of overexpressing chick
G
on the coupling of the M
receptor to
GIRK1. Even with the overexpression of chick G
,
carbamylcholine did not stimulate M
mAChR-sensitive
inward-rectifying K
currents. Furthermore,
co-expression of M
mAChR and GIRK1 with G
had no effect on either the magnitude or characteristics of these
carbamylcholine-stimulated Cl
currents.
To
determine whether the M mAChR coupled to GIRK1, M
and GIRK1 were co-expressed in X. laevis oocytes.
Carbamylcholine (10 µM) evoked
Ba
-sensitive currents of 298 ± 29 nA (Table 1) that were decreased by 99% to 4 ± 4 nA (n = 5, p < 0.05) in response to pretreatment with
pertussis toxin. The magnitude of this current was not significantly
different from that observed in oocytes expressing M
and
GIRK1 (Table 1). In cells expressing the M
receptor
alone, no significant current could be detected in response to
carbamylcholine. The current-voltage relationship of carbamylcholine
stimulation of oocytes co-expressing GIRK1-M
mAChR
demonstrated a strong inward rectification (Fig. 3I)
characteristic of GIRK1 currents.
As seen with M mAChR,
carbamylcholine stimulation of oocytes co-expressing CIR, M
mAChR, and GIRK1 was 13-fold higher than in the absence of CIR ( Table 1and Fig. 3H) and had no effect on the
shape of the current-voltage relationship (Fig. 3I).
Pertussis toxin reduced the carbamylcholine-stimulated current in
oocytes expressing CIR and GIRK1 by 75% from 4010 ± 803 nA to
544 ± 170 nA (n = 9, p < 0.05).
Furthermore the magnitude of the carbamylcholine-stimulated current was
not significantly different from that stimulated in oocytes expressing
the M
mAChR, CIR, and GIRK1 (Table 1). As in the case
of the M
mAChR, overexpression of G
with
M
and GIRK1 with or without CIR had no effect on the
magnitude or characteristics of the carbamylcholine-stimulated
currents.
Others have demonstrated the ability of M mAChR to activate Ca
-stimulated Cl
currents in oocytes(22) . We observed in some, but not
all oocytes co-expressing M
or M
mAChR with
GIRK1, small oscillatory spike-like currents superimposed on the
carbamylcholine-stimulated K
currents (Fig. 3J). It has been suggested that these spike-like
currents represent the weak activation of endogenous oocyte
Cl
channels(23, 24) .
In order to
determine whether expression of chick G might enhance
PI coupling of M
or M
to these
Ca
-activated Cl
channels, we
assessed the characteristics of carbamylcholine-stimulated currents in
oocytes expressing chick G
. In oocytes expressing
chick G
with either M
or M
mAChR and GIRK1, we observed no enhancement of coupling to the
Ca
-activated Cl
currents.
These data demonstrate that we have cloned a G-protein-gated
inward-rectifying K channel cDNA from chick with a
high degree of identity to the rat GIRK1. The mRNA coding for this
channel is expressed in brain, atrium, and at surprisingly high levels
in ventricle. The channel is coupled to M
and M
isoforms of the muscarinic receptor via a pertussis
toxin-sensitive G-protein, but not to the M
receptor. When
M
and M
receptors and GIRK1 are co-expressed
with CIR in oocytes, the carbamylcholine-stimulated current increased
markedly without affecting the specificity of receptor-effector
coupling.
Recently using COOH-terminal deletion mutants of rat
GIRK1, Reuveny et al.(25) found that the COOH
terminus was involved in activation by . Truncation of GIRK1
at Leu-403 or Pro-355 resulted in the loss of response in oocytes
expressing G
and G
. Takao et
al.(26) made chimeras of GIRK1 and the inward-rectifying
K
channel (IRK1) in which the COOH-terminal segment of
IRK1 (residues 261-428) and GIRK1 (residues 339-401) were
exchanged. Only the chimera expressing the carboxyl terminus of GIRK1
was responsive to muscarinic stimulation. Studies with inside-out
patches from rat atrial myocytes suggested that six different
combinations of recombinant
subunits were capable of
activating the inwardly rectifying potassium channel(11) . The
present data suggest that differences in amino acid sequences between
chick and rat GIRK1 had no effect on the characteristics of
carbamylcholine-M
mAChR-stimulated currents (20) or
the ability of CIR to interact with GIRK1(12) , leading to an
increase in carbamylcholine induced K
currents.
The
novel finding in the present study is that the M muscarinic
receptor is coupled to GIRK1. This demonstrates that M
and
M
are coupled to similar effectors in all cases so far
examined. This finding, coupled with the previous observation that
M
mRNA is found in high levels in the brain (5, 27) and that M
receptor protein is
expressed throughout the brain, but at high levels in the basal
ganglia(3, 4) , suggest that in brain muscarinic
agonist activation of M
may function at least in part
through stimulation of this inward-rectifying K
channel.
Although subunits have been implicated in
the activation of a number of effector systems, including
-adrenergic receptor kinase, phospholipase C, certain isoforms of
adenylate cyclase, and mitogen-activated protein kinase (for review,
see (28) ), factors that determine the specificity of
released following agonist receptor binding for activation
of a specific effector system are not well understood. Although no
direct studies of the role of
in activating the
Cl
channel or GIRK1 are presented here, experiments
described here do demonstrate that three different isoforms of the
muscarinic receptor all of which are known to mediate the release of
following agonist binding have a very high degree of
specificity for activation of either GIRK1 or the
Ca
-stimulated Cl
channel in Xenopus oocytes. Furthermore, expression of chick
G
with the M
receptor had no effect on
coupling of M
to GIRK1, and expression of chick
G
with the M
receptor had no effect on the
low level of coupling of M
mAChR to Cl
channel activation. Although these studies do not rule out the
possibility that levels of
may be limiting in the oocyte,
the large size of the M
-activated Cl
currents should be sufficient to affect activation of GIRK1 if
such coupling existed. Since in vitro studies suggest that
various
subunits can activate GIRK1, it seems unlikely that
the specific
subunits expressed in oocytes can account for
the lack of coupling of M
mAChR to GIRK1. Other factors
such as compartmentalization of
released in response to
receptor activation or precoupling of receptor G-protein and effectors
may account for the remarkable specificity of receptor GIRK1 coupling.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L35555[GenBank].