From the Howard Hughes Medical Institute, Childrens' Hospital, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The cardiac G protein-gated K+
channel, IKACh, is directly activated by G protein
subunits (G
). IKAChis composed of two
inward rectifier K+ channel subunits, GIRK1 and GIRK4.
G
binds to both GIRK1 and GIRK4 subunits of the
heteromultimeric IKACh. Here we delineate the
G
binding regions of IKACh by studying direct G
interaction with native purified
IKACh, competition of this interaction with peptides
derived from GIRK1 or GIRK4 amino acid sequences, mutational analysis
of regions implicated in G
binding, and functional
expression of mutated subunits in mammalian cells. Only two GIRK4
peptides, containing amino acids 209-225 or 226-245, effectively
competed for G
binding. A single point mutation
introduced into GIRK4 at position 216 (C216T) dramatically reduced the
potency of the peptide in inhibiting G
binding and
G
activation of expressed GIRK1/GIRK4(C216T)
channels. Conversion of 5 amino acids in GIRK4 (226-245) to the
corresponding amino acids found in the G protein-insensitive IRK1
channel, completely abolished peptide inhibition of G
binding to IKACh and G
activation of
GIRK1/mutant GIRK4 channels. We conclude from this data that
G
binding to GIRK4 is critical for IKACh
activation.
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INTRODUCTION |
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Acetylcholine (ACh)1
secreted from the vagus nerve binds cardiac muscarinic receptors,
initiating a sequence of events leading to slowing of heart rate
(1-3). IKACh, an inwardly rectifying, K+-selective channel, mediates part of this process by
hyperpolarizing pacemaker cells in sinoatrial and atrioventricular
nodes of the heart (for review, see Ref. 4). Muscarinic, adenosine, and other receptors (5) all catalyze the release of G subunits from pertussis toxin-sensitive heterotrimeric G proteins, which in turn directly activates the channel (6-9). Cardiac
IKACh is composed of two homologous inward rectifier
K+ channel subunits, GIRK1 (9, 10) and GIRK4 (11), which form a heterotetramer consisting of two GIRK1 and two GIRK4 subunits (12). Similar complexes comprised of GIRK1 and GIRK2 (13-17), or GIRK1
and GIRK3 (13) appear to form neuronal G protein-gated K+
channels. Both channel subunits bind G
with similar affinity (18-22).
An important finding that is crucial to understanding
G regulation of IKACh is that
G
activates homomultimeric GIRK4 (11, 15) or
homomultimeric GIRK2 (16) channels. This implies that the GIRK4 subunit
alone contains the necessary elements required for activation. In
contrast to GIRK2 and GIRK4, GIRK1 subunits are not functionally
expressed as homomultimers (23-25), and this fact limits conclusions
that can be drawn about GIRK1 contributions to G
regulation.
Initially, GIRK1 was thought to comprise the entire IKACh
channel (9, 10), and initial studies on the mechanism of
G activation of IKACh focused on GIRK1.
Chimeric channels were constructed from GIRK1 and non-G
protein-sensitive K+ channel inward rectifier (Kir)
subunits to examine this problem. Results from the expression of these
chimeras in Xenopus oocytes were used to conclude that the
GIRK1 C-terminal and/or N-terminal cytoplasmic domains (18, 26-28)
conferred G
sensitivity on the channel complex.
However, expression studies in Xenopus oocytes are
complicated by the fact that GIRK1 forms a functional channel complex
only by combining with an intrinsic oocyte GIRK4 homolog (XIR; Ref.
23). It should be noted that chimeras containing structural elements
that promote interaction with XIR can be confused with elements
responsible for G
regulation. Also, a consistent
shortcoming of previous chimeric approaches has been the lack of direct
biochemical determination of chimeric expression levels, plasma
membrane targeting, and heterotetramer formation.
Several biochemical studies attempted to identify a G
binding site within channel subunits. These studies usually employed
G
binding to glutathione
S-transferase-fused GIRK1 fragments and demonstrated that
G
bound fusion proteins containing regions of GIRK1
cytoplasmic N-terminal and/or C-terminal domains (18, 19, 21, 22).
Here we localize G binding sites on the GIRK4 subunit
of native cardiac IKACh using an in vitro
G
binding assay and competition of binding with
peptides derived from the subunit primary structure. We confirmed the
functional significance of these binding sites using mutational
analysis of regions implicated in G
binding and
functional expression of mutated subunits in mammalian cells. We
identified a single GIRK4 cysteine residue important for
G
binding and demonstrated that G
binding to GIRK4 is critical for channel activation.
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MATERIALS AND METHODS |
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Protein Purification, G Binding, and Peptide
Preparation--
G proteins were isolated from bovine brain, separated
into G
and G
subunits as described
(29) and additionally purified by affinity chromatography over
immobilized G
(30). Bovine atrial plasma membranes were
isolated as described (31). Membranes were solubilized in 1.0%
CHAPS-HEDN buffer (in mM: 10 HEPES, 1 EDTA, 1 dithiothreitol, and 100 NaCl) containing protease inhibitors and
IKACh was purified by immunoprecipitation (12, 32).
Immunoprecipitates were washed with solution containing 0.1%
CHAPS-HEDN buffer and used for the G
binding assay (20). 20 amino acid length peptides derived from GIRK1 and GIRK4 amino
acid sequences were synthesized in the Mayo Protein Core Facility (Mayo
Clinic, Rochester, MN), high pressure liquid chromatography-purified, and their identity and purity verified by mass spectrometry. Peptide N
and C termini were carboxymethylated or
-amidated, respectively. For
competitive binding experiments, peptides were dialyzed against H2O for 48 h, their concentration measured by
spectrophotometry (optical density at 205 nm), and the peptides were
transferred into the binding assay solution. G
was
preincubated with peptides for 20 min before incubation with the
channel protein. G
was labeled with 125I
using the Bolton-Hunter reagent (NEN Life Science Products) as
described (20).
Cell Culture and Transfection-- CHO and COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Sigma) at 37 °C, 5% CO2. Cells were plated at 3 × 106 or 2 × 106 cells/100-mm dish, respectively, 1 day prior to transfection. For electrophysiology experiments in CHO or COS-7 cells, each 100-mm plate was transfected with 5 µg of plasmid DNA, (2 µg of each plasmid containing either wild type or mutant GIRK1 or GIRK4 cDNA and 1 µg of pGREEN lantern-GFP (Life Technologies, Inc.) using TransIT LT-1 lipid transfection reagent (PanVera Corp, Madison, WI). For biochemical and immunofluorescence detection experiments in COS-7 cells, cells were transfected with 5 µg each of plasmids containing wild type and/or mutant subunit using TransIT LT-1 reagent (PanVera Corp, Madison, WI).
Site-directed Mutagenesis, Immunoflourescence Detection, and Subunit Coimmunoprecipitation-- Deletion and point mutants of GIRK1 and GIRK4 and the extracellular epitope-tagged GIRK1-Flag construct were generated by oligo-mediated mutagenesis using the site elimination method (Transformer Mutagenesis, CLONTECH). The pCDNA3 vector carrying the coding region of either C-terminally tagged GIRK1 (GIRK1-AU5) or GIRK4 (GIRK4-AU1) was used as a template for mutagenesis. Mutations were verified by DNA sequencing. Mutant subunits were evaluated for expression levels, cellular localization, and subunit interaction after transient expression in COS-7 cells as described (24). Triton X-100 was omitted in experiments designed to detect GIRK1-Flag cell surface expression.
Electrophysiological Analysis--
Transfected cells were
visualized using an inverted fluorescence microscope to detect GFP
expression. Electrophysiological recordings were performed from CHO-K1
and COS-7 cells using the patch clamp technique in the inside-out
configuration. Pipette and bath solutions were identical and contained
(in mM): 140 KCl, 5 EGTA, 10 HEPES-KOH, 2.0 MgCl2, pH 7.2. The resistance of the recording borosilicate
glass pipettes was 2-4 megaohms. Single-channel currents were recorded
at a holding potential of 80 mV using an Axopatch 200B amplifier
(Axon Instruments, Inc.). Data were filtered at 5 kHz, digitized at 20 kHz through a 1200 DigiData A-D converter using pClamp 6 software (Axon
Instruments, Inc.), and stored on disk for off-line analysis. The
channel opening probability was calculated using pClamp 6 software
(Axon Instruments, Inc.) from 5-s segments of inside-out patch
recordings (33). Changes in concentrations of G
used
in the dose-response experiments were achieved by transferring
inside-out patches from the recording chamber to the
G
-containing solution.
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RESULTS |
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To find the GIRK subunit locus responsible for G
binding to IKACh, we first studied in vitro
binding of purified G
to purified cardiac
IKACh. This approach included using peptides corresponding
to stretches of the channel subunits in a competition binding assay.
The cytoplasmic domains of GIRK4 and cytoplasmic domains of GIRK1 not
strongly homologous to GIRK4, were screened by dividing them into
contiguous nonoverlapping peptides of ~20-amino acid length. Our
strategy assumed that peptides that correspond to
G
-binding epitopes would interfere with the
interaction of G
with GIRK channel subunits. Not
every region of the GIRK subunit was examined. Peptides corresponding to transmembrane domains of GIRK were not used because it seemed unlikely that G
would interact with the channel's
transmembrane domains. Also, we did not test amino acids 18-32 of
GIRK4 because they comprise the epitope for the immunoprecipitating
antibody, and would interfere with the binding assay. Finally, some
synthesized peptides were insoluble and could not be evaluated. A
summary of peptide influence on G
binding is shown in
Fig. 1.
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Almost all peptides tested at concentrations of 50-200
µM significantly inhibited G binding to
purified IKACh. However, inhibition was not consistent,
varying from batch to batch of peptide. After peptides were dialyzed
against distilled water, most peptides did not inhibit binding at
concentrations less than 200 µM, but all weakly inhibited
binding at higher concentrations. The simplest interpretation of this
result was that peptide sample impurities inhibited G
interaction with the channel and that most of these impurities were
removed by dialysis. All peptides we tested corresponding to the N
terminus of both GIRK1 and GIRK4 were ineffective. Only four of the
peptides tested against the C terminus inhibited in vitro
G
binding to the channel in a
concentration-dependent manner at concentrations
significantly less than 200 µM (Fig.
2). The specificity of this inhibition was verified in experiments with scrambled and truncated peptides (Figs. 3,
4, and 6).
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The GIRK1(364-383) peptide (Fig. 2) potently inhibited
G binding to native IKACh. Truncation of
only 4 amino acids from this peptide (amino acids 364-367; MLLM)
resulted in its complete inactivation (Fig. 3B), leading us
to conclude that amino acids 364-367 in GIRK1 participated in
G
binding to IKACh. However,
heteromultimeric channels containing the mutant GIRK1 subunit in which
amino acids 364-367 had been deleted were activated by
G
indistinguishably from wild type GIRK1/GIRK4 channels (Fig. 3C). Because the peptide competition result
suggested that the MLLM sequence was critical for inhibition of
G
binding to the channel complex, and deletion of
these amino acids from GIRK1 did not change channel activation, it is
possible that GIRK1(364-383) inhibited IKACh activation by
competition with a G
-binding domain on GIRK4. Indeed
GIRK1(364-383) significantly inhibited G
binding to
the recombinant GIRK4 subunit, which was expressed and purified from
COS-7 cells. 6 of the 8 amino acids of the 268-275 stretch of GIRK4
were homologous to this region of GIRK1 (5/8 identical). Unfortunately
we were not able to directly test this site for G
binding using peptide blockade because the GIRK4 peptide derived from
this area was insoluble. The region from GIRK1 (364-383) also contains
a motif previously implicated in G
activation of
IKACh. Chen et al. (34) suggested that the
putative G
binding region (motif
(Q/N)XXER) of adenylyl cyclase type 2 (AC2 (956-982); Fig.
3A) had homology to GIRK1(378-382). Indeed, their
experiments showed that AC2 (956-982) peptide blocked cardiac
IKACh activation in excised inside-out patches. We assessed
the functional role of GIRK1(378-382) by examination of whether
AC2 (956-966) and homologous GIRK1(378-387) peptides inhibited
G
binding to IKACh but failed to detect
any inhibition (Fig. 3B). We also mutated and deleted the
putative G
binding motif NSKER (GIRK1 378-382) and
coexpressed the mutant GIRK1 with GIRK4 in COS-7 cells but found no
difference in activation by G
(n = 4;
data not shown).
As shown in Fig. 2, the most potent inhibitor of G
binding to IKACh was the GIRK4 (209-225) peptide. This
peptide corresponded to the carboxyl-terminal region of GIRK4, lying
just outside the second putative transmembrane domain (Fig. 1). GIRK4 (209-225) completely inhibited G
binding at
concentrations of 3-5 µM. Deletion of amino acids
209-225 of the GIRK4 subunit (mutant GIRK4(
209-225)) and
expression with wild type GIRK1 abolished G
activation (Fig. 5C). However we failed to detect significant coimmunoprecipitation of GIRK1 with mutant
GIRK4(
209-225) nor did GIRK4(
209-225) promote detectable cell
surface localization of GIRK1 (Fig. 7) characteristic for wild type
GIRK4 (24). The simplest interpretation of these data is that the
region 209-225 in GIRK4 is critical for both G
binding and subunit interaction.
To refine the area responsible for the peptide inhibition, we compared
the sequence of this peptide to homologous sequences of related
G binding GIRK subunits, as well as to corresponding sequences of the G
-insensitive IRK1 (Fig.
4A). Comparative analysis suggested that residues Ser-209,
Phe-219, or Asp-224 might be involved in the interaction with
G
. Mutated GIRK4 peptide (F219W) and homologous
peptides derived from GIRK1(203-219) and IRK1(202-218) were then used
to test the significance of these amino acids in G
binding. Surprisingly, the IRK1(202-218) peptide inhibited
G
binding as well as the peptide GIRK4(209-225; Fig.
4B). Also, the GIRK1-derived peptide, GIRK1(203-219), did not inhibit G
binding at concentrations up to 50 µM (Fig. 4B). Sequence comparison suggests
that the substitution of Cys-216 in GIRK4 for Thr at position 210 in
the homologous GIRK1 peptide might be responsible for the observed
difference in potency of inhibition between GIRK4 and GIRK1 peptides.
To test this possibility, the GIRK4 (C216T) mutant subunit was
coexpressed in CHO cells with GIRK1. As for COS-7 cells, CHO cells do
not contain endogenous GIRK-like proteins (tested with GIRK-specific antibodies; data not shown) and do not support functional expression of
GIRK1(11). Heteromultimeric channels containing the GIRK4(C216T) mutant
produced functionally active IKACh channels but with a greater than 50-fold lower EC50 for G
(Fig. 5, A and C).
The rightward shift in potency of G
activation was not because of changes in either surface localization or
heteromultimerization with GIRK1 (Fig. 7), confirming that Cys-216 in
GIRK4 is important for binding of G
to
IKACh.
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A peptide designed from an adjacent region of GIRK4, GIRK4(226-245),
was also found to significantly inhibit G binding to
IKACh(Fig. 2). Again, sequence comparison between other
G
binding GIRK subunits and the homologous, but G
protein-independent inward rectifier, IRK1, led us to evaluate 5 amino
acids potentially participating in G
binding (Fig.
6A). An IRK1 peptide amino
acids 219-238, homologous to GIRK4(226-245), did not inhibit G
binding to IKACh. Mutation of these 5 amino acids in GIRK4 to the corresponding amino acids in IRK1 ablated
G
activation after coexpression of this mutant GIRK4
with GIRK1 (Fig. 6B). These specific GIRK4 mutations did not
influence assembly with GIRK1 or targeting to the plasma membrane (Fig.
7). These data support the hypothesis
that the GIRK4(226-245) locus is also important for the
G
binding and activation.
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Finally, we tested a peptide derived from the GIRK1 region (220-239)
homologous to GIRK4(226-245), which contained amino acids conserved in
all GIRKs. The peptide inhibited G binding to
IKACh (Fig. 2) with a dose response similar to
GIRK4(226-245). This result was not surprising because this region is
highly conserved between GIRK4 and GIRK1. We suggest that the
GIRK1(220-239) peptide also inhibited G
binding to
the previously identified GIRK4(226-245). However, we cannot rule out
the possibility that this region of GIRK1 is also important for
interaction of channel with G
.
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DISCUSSION |
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We have combined peptide competition and mutagenesis techniques to
delineate domains within GIRK1 and GIRK4 that bind G and play a role in G
-mediated activation of
GIRK1/GIRK4 channels. In summary, we have identified a single GIRK4
region (amino acids 209-245) that appears to be crucial for
G
binding. This region lies close to the putative
second transmembrane domain of GIRK4.
We have identified several peptides derived from both GIRK1 and GIRK4
that block G binding to immunopurified cardiac IKACh. In confirmation of these results, the mutation of
regions that bound G
also disrupted
G
-mediated channel activation. Deletion and point
mutations in regions corresponding to blocking peptides from GIRK4
either completely disrupted or significantly reduced the potency of
G
in activating GIRK1/mutant GIRK4 channels measured
in inside-out patches exposed to purified G
subunits.
Interestingly, deletion of GIRK1 amino acids corresponding to the
peptide capable of inhibiting G
binding to
IKACh had no effect on G
activation of
GIRK1(
364-367)/GIRK4 channels. This result suggests that either the
GIRK1 peptide inhibited binding to homologous sites on the GIRK4
subunit or that G
binding to the GIRK1 site was not
necessary for channel activation. The observation that amino acids
273-462 of GIRK1 bound G
subunits (19) supports our
hypothesis that G
binding to GIRK1 is not required for channel activation. We found that Cys-216 in GIRK4 is important for
high affinity binding of G
to the native channel. This same position is occupied by Thr in GIRK1. Because mutation of
Cys-216 in GIRK4 to threonine dramatically reduced the potency of
G
, we suggest that G
does not bind
to this region of GIRK1. Combined with the known
G
-dependent activation of GIRK4
homomultimers, we suggest that GIRK4 plays a necessary role in the
G
-dependent activation of
heteromultimeric IKACh.
Huang et al. (19) showed that a peptide corresponding to
amino acids 434-462 of GIRK1 inhibited single channel activity in
oocytes expressing GIRK1 and G or GIRK1/GIRK4 and G
. In contrast, we found that peptides derived from this region of GIRK1 had no effect on G
binding.
Furthermore truncation of GIRK1 at amino acid 373 and coexpression with
GIRK4 in COS-7 cells did not alter channel
acitivation.2 Although Huang
et al. (22) found a role for N-terminal cytoplasmic domain
(34-86 in GIRK1) binding to G
we could not confirm this result because peptides corresponding to these regions of GIRK1(67-83) or GIRK4(66-85) were insoluble and could not be tested in our binding assay.
We found that some peptides, which did not influence
G binding to the channel, nevertheless suppressed
channel activation by G
when applied to inside-out
patches. A reasonable explanation of these results is that the peptides
interacted with the channel itself and interfered with intersubunit
interaction. Such an explanation is supported indirectly by
noncompetitive inhibition of the GIRK1/XIR channel by a C-terminal
GIRK1 peptide (35). Similarly, the fact that an IRK1 peptide was also
found to block G
binding to IKACh does
not imply that IRK1 has a G
binding site, although
this possibility cannot be excluded.
In conclusion, G binding to IKACh
channels involves a region of the carboxyl tail of GIRK4 in close
proximity to the putative second transmembrane domain, and binding of
G
to this region is critical for channel activation.
Interestingly, these regions lie close to the putative pore domain of
the tetrameric complex, perhaps providing an important clue as to why
the weaver mouse GIRK2 pore mutant (a close relative of
GIRK4) demonstrated loss of G
sensitivity (16).
Although we have shown that G
binds to GIRK1, our
attempts to demonstrate that this binding plays a role in
G
-induced channel activation were not successful.
However, it is likely that a combined GIRK1/GIRK4 conformation
determines the G
binding site, and activation cannot
be fully understood in the absence of detailed structural data.
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FOOTNOTES |
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* This work was supported by National Institutes of Health, National Heart, Lung, and Blood Institute Grant 54873 (to D. E. C.), National Research Service Award/Howard Hughes Medical Institute (to M. K.), the Mayo Foundation (to J. N.), and the Howard Hughes Medical Institute (to D. E. C.).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: Howard Hughes Medical
Institute, Professor of Neurobiology, Pediatrics Childrens' Hospital,
Harvard Medical School, 1309 Enders, 320 Longwood Ave., Boston, MA
02115. Tel.: 617-355-6163; Fax: 617-730-0692; E-mail: clapham{at}rascal.med.harvard.edu.
1 The abbreviations used are: ACh, acetylcholine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CHO, Chinese hamster ovary.
2 M. E. Kennedy, unpublished data.
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REFERENCES |
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