G-protein-coupled inwardly rectifying
K+ (GIRK; Kir3.x) channels are the primary
effectors of numerous G-protein-coupled receptors. GIRK channels
decrease cellular excitability by hyperpolarizing the membrane
potential in cardiac cells, neurons, and secretory cells. Although
direct regulation of GIRKs by the heterotrimeric G-protein subunit
G
has been extensively studied, little is known about the number
of G
binding sites per channel. Here we demonstrate that purified
GIRK (Kir 3.x) tetramers can be chemically cross-linked to exogenously
purified G
subunits. The observed laddering pattern of G
attachment to GIRK4 homotetramers was consistent with the binding of
one, two, three, or four G
molecules per channel tetramer. The
fraction of channels chemically cross-linked to four G
molecules
increased with increasing G
concentrations and approached
saturation. These results suggest that GIRK tetrameric channels have
four G
binding sites. Thus, GIRK (Kir 3.x) channels, like
the distantly related cyclic nucleotide-gated channels, are tetramers
and exhibit a 1:1 subunit/ligand binding stoichiometry.
 |
INTRODUCTION |
Roughly 2% of the human genome encodes G-protein-coupled
receptors (1). Agonist binding to these G-protein-coupled receptors catalyzes the activation of G
and G
subunits of heterotrimeric G-proteins. The free G
and G
subunits can then interact
independently or in concert with numerous effectors. G
regulates
processes as diverse as the yeast pheromone response (2-4) and
mammalian heart rate (5). The increasing list of G
effectors
includes ion channels (6-12), phospholipase C
(13), adenylyl
cyclases (14), G-protein-coupled receptor kinases (15),
PI1 3-kinase (16), plasma
membrane Ca2+ pumps (17), Bruton's tyrosine kinase (18),
and calmodulin (19). Little is known about how G
interacts with
its effectors. The repeating WD40 motif of G
gives it a rigid
propeller-like structure, which does not appear to be altered upon its
interaction with effectors (20-23).
Homotetrameric and heterotetrameric combinations of the four known
mammalian GIRK subunits are activated by neurotransmitters in the
nervous system, pancreas, and heart. Muscarinic (m2, m4),
-aminobutyric acid (GABAB),
D2-dopamine,
2-adrenergic, opiate, somatostatin, and adenosine all employ the G
i-G
signal transduction system to activate GIRK channels via direct G
binding to the tetrameric channel. GIRK4-knockout mice have
irregularities in heart rate variability (5) and difficulties with
spatial learning (24). GIRK2-knockout mice are prone to seizures (25).
Weaver mice have a mutation in the pore domain of the GIRK2
subunit (26) that renders the channel nonselective (27) and results in
the degeneration of cerebellar granule cells (28) and the dopaminergic neurons of the substantia nigra (29, 30).
The native atrial IKACh channel is composed of two GIRK1
subunits and two GIRK4 subunits (31-33) that comprise a channel that mediates neuronal regulation of heart rate. Biochemical studies indicate that G
binds the native IKACh complex with a
Kd of 55 nM (9). G
binds both
recombinant GIRK1 (Kd = 125 nM) and
GIRK4 (Kd = 50 nM) (9). GIRK1 subunits are unable to form functional homomultimers (34), whereas GIRK4 homomultimers have been biochemically isolated from bovine atria (35).
GIRK2/3 and GIRK1/2 heteromultimers have also been isolated from brain
(1, 36). The C-terminal tail of GIRK1 and GIRK4 subunits bind G
(9, 32, and 37-46), but the detailed steps of how this binding leads
to channel gating is not known. Furthermore, there is limited data
about the areas of G
that bind GIRK channel subunits (43, 47) and
about how many G
subunits can bind the tetrameric channel complex.
We have used a biochemical approach to determine how many G
subunits bind GIRK tetramers. By extending our previous chemical cross-linking studies (31), which indicated that GIRKs form tetramers,
we demonstrate that GIRK4 homotetramers bind four G
subunits in
their natural membrane environment.
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EXPERIMENTAL PROCEDURES |
Isolation, Solubilization, and Purification of GIRK1/GIRK4
Heteromultimers (IKACh) from Native Atrial
Membranes--
Bovine atrial plasma membranes were isolated (48) and
solubilized as described (31). Native GIRK1/GIRK4 heteromultimers (IKACh) were purified to greater than 90% homogeneity as
described (31). The protease inhibitors leupeptin (50 µg/ml
Sigma-Aldrich Inc.), phenylmethylsulfonyl fluoride (100 µg/ml,
Sigma-Aldrich Inc.), aprotinin (1 µg/ml, Sigma-Aldrich Inc.), and
pepstatin (2 µg/ml, Sigma-Aldrich Inc.) were used during all steps of
the purification.
Expression and Isolation of GIRKs from COS7 and CHO
Cells--
Plasma membrane proteins containing GIRK1-AU5 and GIRK4-AU1
were isolated from COS7 cells and solubilized as described (49).
G
Purification--
G-proteins were isolated from bovine
brain and separated into G
and G
subunits (50) and were
further purified by affinity chromatography using immobilized G
(51).
G
Binding in Membranes--
Isolated COS7 cells or native
atrial membranes were treated for 1 h with 100 mM
dithiothreitol and then dialyzed against 20-50 mM HEPES,
100 mM NaCl, pH 7.4-7.5 (G
binding buffer).
Individual membrane aliquots were preincubated with purified bovine
brain G
and rotated for 20 min at room temperature prior to
cross-linking. The G
stock solution was in G
binding buffer
containing 0.1% CHAPS (0.1% G
binding buffer). The final CHAPS
concentration was less than or equal to 0.1%.
G
Binding to Solubilized Protein--
Solubilized COS7
membrane proteins were treated for 1 h with 100 mM
dithiothreitol and then dialyzed against 0.1% G
binding buffer.
Individual aliquots were preincubated with purified brain G
(supplied in 0.1% G
binding buffer) and rotated for 20 min at
room temperature prior to cross-linking.
Chemical Cross-linking--
5 mM
dithiobis[sulfosuccinimidylpropionate] (DTSSP, Pierce Chemical,
Rockford, IL) was prepared as an 11× stock solution immediately prior
to use in 100 mM HEPES-containing buffer, pH 7.5. Iodine was added only to solutions containing purified IKACh.
Cross-linking reactions were allowed to proceed for 30 min at room
temperature and then quenched with 50 mM Tris. Typically
5-10 µg of membrane proteins were used per reaction in a final
volume of 15 µl.
SDS-PAGE and Immunoblotting--
Atrial membrane proteins or
recombinant GIRK proteins were resuspended in Laemmli sample buffer
containing 100 mM dithiothreitol (or 30 mM
iodoacetamide when a cross-linking agent was used) for 15 min at
50 °C, 30 min at room temperature, and 15 min at 50 °C. 3-10%
separating, 3% stacking, and pre-cast 2-15% (ISS) gels were
utilized. Samples were analyzed by immunoblotting with anti-GIRK4 antibodies (generated against amino acids 19-32, Ref. 31) and/or anti-GIRK1 antibodies (generated against the last 156 amino acids of
GIRK1, Ref. 31). Several antibodies were tested for use in the
anti-G
immunoblotting experiments. Only one anti-G
antibody (anti-KTREGNVRVSREL, Chemicon International, Inc. Temecula, CA) reacted
with G
after DTSSP treatment. Typically, DTSSP treatment reduced
total antigenic signal by >90%, >60%, and >95% for anti-GIRK4, anti-GIRK1, and anti-G
antibodies, respectively. Transfer times for immunoblot analysis were extended to >2 h at 15 V to improve transfer of the high molecular weight complexes. A GS-700 imaging densitometer (Bio-Rad Laboratories, Hercules, California) was used to
analyze the protein gels and immunoblots. Molecular masses were
calculated using densitometry profiles from a combination of prestained
high molecular mass markers (Bio-Rad) and low and high molecular mass
markers (Amersham Pharmacia Biotech). In a portion of the gels,
thyroglobulin (Amersham Pharmacia Biotech) was added to ensure
linearity up to 330 kDa.
 |
RESULTS |
Previous chemical cross-linking studies demonstrated that GIRK
subunits form tetrameric channels and that the native atrial channel
IKACh is composed of 2 GIRK1 and 2 GIRK4 subunits (31). Complete cross-linking of purified atrial IKACh formed a
single adduct with a total molecular mass that was most consistent with a tetramer. In addition, partial cross-linking of purified
IKACh produced subsets of molecular weight adducts
consistent with monomers, dimers, trimers, and tetramers. In this
study, we extended our previous experiments to determine how many
G
molecules can be cross-linked to GIRK tetramers.
G
Cross-linking to Purified Native IKACh--
To
test whether GIRK1/GIRK4 heteromultimers could be directly and
specifically cross-linked to G
, we used isolated native atrial
GIRK1 and GIRK4 subunits (31) and bovine brain G
(9). Isolated
GIRK1 and GIRK4 heterotetramers were preincubated with isolated
G
, followed by cross-linking with DTSSP (Fig.
1). Although the predicted molecular
masses of GIRK1 and GIRK4 subunits are 56 and 45 kDa, respectively, the
glycosylated GIRK1 migrates in a broad band between 67-72 kDa (9). In
the absence of G
, a band formed at 230 kDa, consistent with the
total molecular mass of two GIRK1 (56, 67-72 kDa) and two GIRK4 (45 kDa) subunits. In the presence of G
, a band corresponding to a
molecular mass of 390 kDa was detected. Because G
, GIRK1
and GIRK4 were the predominant proteins present, we interpreted the
160-kDa shift as the result of direct cross-linking of G
to GIRK
channels. The molecular mass of G
is 42 kDa, suggesting that the
160-kDa shift was because of cross-linking of several G
molecules
to the GIRK1/GIRK4 heterotetramers. Because GIRK4 can form
homotetramers, we repeated the previous cross-linking experiment using
recombinant GIRK4 subunits. In the absence of G
, cross-linking of
recombinant GIRK4 resulted in a band at 170 kDa, corresponding to GIRK4
homotetramers. When G
was added to recombinant GIRK4,
cross-linking yielded a band at ~320 kDa (not shown). This banding
pattern is most consistent with four specific and saturable G
binding sites per GIRK4 homotetramer.

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Fig. 1.
Purified native atrial GIRK1 and GIRK4
heterotetramers can be directly and specifically cross-linked to
purified G . A,
schematic depicting procedure used to generate B. Squares represent GIRK subunits. B, 2-15%
SDS-PAGE and anti-GIRK1 immunoblotting of solubilized GIRK1 and GIRK4
heterotetramers. Lane 1, pure solubilized native atrial
GIRK1 and GIRK4 heterotetramers treated with 1 mM DTSSP.
Lane 2, pure solubilized GIRK1 and GIRK4 heterotetramers
preincubated with 3 µM G and treated with 1 mM DTSSP. Exposure times for lanes 1 and
2 were not identical.
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Cross-linking of Membrane-confined GIRK4 Homotetramers--
It is
important to study GIRK-G
binding in its membrane environment
because phosphatidylinositol bisphosphate (PIP2) (52, 53)
is involved in the G
-mediated activation of GIRK channels. Our
previous GIRK cross-linking studies employed isolated solubilized channels (31). In this study, we tested whether GIRK subunits could be
cross-linked into tetramers in membranes. After DTSSP cross-linking of
membranes from COS7 cells expressing either recombinant GIRK4, GIRK1,
or GIRK1 and GIRK4, SDS-PAGE yielded 180-220-kDa bands (Fig.
2B, lanes 1-3).
These bands are similar in molecular mass to those produced when
solubilized GIRKs are cross-linked into tetramers (31). Of the GIRK
tetramers, the chemically cross-linked GIRK4 homotetramers produced the
narrowest band, around 190 kDa (Fig. 2B, lane 1). In
addition, the GIRK4 band cross-linked directly in membranes (Fig.
2B, lane 1) was narrower than that of GIRK4 that had been
solubilized before cross-linking (31).

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Fig. 2.
Membrane-confined GIRK channels can be
completely chemically cross-linked into tetramers. A,
schematic depicting procedure used to generate B. B, CHO membrane proteins were cross-linked with DTSSP and
analyzed by 2-15% SDS-PAGE and immunoblotting. Lanes 1-3,
membranes from CHO cells transfected with the indicated GIRK subunit
and cross-linked with 2.5 mM DTSSP.
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Partial G
Cross-linking to Membrane-confined GIRK
Tetramers--
GIRK4 homotetramers were used in our membrane-confined
GIRK-G
binding experiments because cross-linking of GIRK4
homotetramers in membranes yielded the narrowest bands. We altered our
cross-linking conditions to verify that there were indeed four G
binding sites in the GIRK tetramer. DTSSP and G
concentrations
were adjusted so that variable numbers of G
molecules were
cross-linked to the GIRK4 homotetramers. COS7 cells were transiently
transfected with GIRK4, and their membranes were divided into separate
aliquots. Each aliquot was treated with variable amounts of G
and
DTSSP and then analyzed by SDS-PAGE and immunoblotting. Untreated
GIRK4, migrated as a 47-kDa trichloroacetic acid-disruptable monomer (Fig. 3B, lane 1).
GIRK4 cross-linked with DTSSP migrated as a 170-kDa tetramer (Fig.
3B, lane 2). GIRK4, preincubated with G
and
then cross-linked with DTSSP, resulted in a laddering pattern of four
main adducts (in addition to the GIRK4 homotetramer adduct) with
consistent 40-45-kDa increments between bands (Fig. 3B,
lanes 3 and 4). The proportion of high molecular
weight adducts increased with increasing G
concentrations. Unlike
our previous experiments that used solubilized GIRK protein, a
population of the membrane-confined GIRK4 homotetramers (166 kDa)
remained resistant to any G
binding. One possible explanation for
this observation is that a subpopulation of GIRK4 homotetramers may not
have been accessible to the exogenously applied G
. In five
independent trials, four GIRK-G
adducts consistently appeared. In
some trials, high molecular mass, lower intensity smears formed, but
these bands were not consistently reproducible. A laddering pattern was
not formed when G
was boiled prior to its addition to membranes
(data not shown). We hypothesize that the five adducts formed by
treatment of G
and GIRK4-containing solutions with DTSSP
represent the binding of zero, one, two, three, and four G
molecules to GIRK4 homotetramers.

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Fig. 3.
DTSSP treatment of membrane-confined GIRK4
homotetramers in the presence of G
creates a laddering pattern consistent with four
G sites per GIRK tetramer.
3-10% SDS-PAGE analysis, anti-GIRK4 immunoblotting and cross-linking
of membranes from COS7 cells that were transfected with GIRK4.
A, schematic depicting procedure used to generate
B. B, lane 1, GIRK4-containing
membranes that were trichloroacetic acid-precipitated prior to
SDS-PAGE. Lane 2, GIRK4-containing membranes cross-linked
with 5 mM DTSSP. Lanes 3 and 4, GIRK4-containing membranes pretreated with 0.13 µM and
0.63 µM G , respectively, followed by cross-linking
with 5 mM DTSSP. Squares represent GIRK
subunits. Circles represent G molecules. The molecular
mass markers at left correspond to panel 1. The
molecular mass markers on the right correspond to adducts in
panel 2. C, GIRK4-containing membranes pretreated
with 3.1 µM G and then cross-linked with 5 mM DTSSP, enlarged view. D, in a separate trial,
densitometry profiles were created by scanning each lane of the gel top
to bottom, which is now represented as top to bottom on the graph.
Profile 1, GIRK4-containing membranes cross-linked with 5 mM DTSSP (0.0 µM G ). Profile
2, GIRK4-containing membranes preincubated with 0.6 µM G and cross-linked with DTSSP (0.6 µM G ). Profile 3, GIRK4-containing
membranes preincubated with 3.1 µM G and
cross-linked with 5 mM DTSSP (3.1 µM
G ).
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Multiple lines of evidence suggest that G
is directly
cross-linked to GIRK channels in our experiments. G
has been
coimmunoprecipitated with GIRK subunits under the conditions used in
our experiments (9). The ~45-kDa increments between cross-linked
GIRK-G
adducts are consistent with the stepwise addition of
42-kDa G
subunits to the channel. Finally, similar results were
obtained even when IKACh and G
were purified to
>95% homogeneity prior to cross-linking. Because, IKAch
and G
are the predominant proteins in solution, the molecular
mass shift with G
addition strongly suggests that G
is
being directly cross-linked to the channel. As a final precaution, we
tested whether the putative GIRK-G
adducts are recognized by
anti-G
antibodies. COS7 cells were transiently transfected with
GIRK4 and their membranes were isolated. The membranes were treated
with G
and DTSSP, followed by SDS-PAGE analysis. Immunoblots were
probed with anti-GIRK4 antibodies then stripped and reprobed with
anti-G
antibodies (Fig. 4,
lanes 1 and 2, respectively). The anti-G
antibodies recognized bands at molecular masses that correspond to the
putative GIRK-G
adducts.

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Fig. 4.
Anti-G
antibodies recognize bands at molecular masses that correspond to
the putative GIRK-G
adducts. 3-10% SDS-PAGE analysis and
immunoblotting of membranes from COS7 cells that were transfected with
GIRK4, pretreated with G , and then treated with DTSSP. Lane
1, GIRK4-containing membranes pretreated with 6.3 µM
G and cross-linked with 4 mM DTSSP probed with
anti-GIRK4 antibodies. Lane 2, (lane 1) stripped
and reprobed with anti-G antibodies.
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|
 |
DISCUSSION |
The present study of G
binding to channel proteins has
several advantages over other approaches. First, we ensured that we
were using intact tetramers throughout our G
binding experiments. In addition, we purposely studied G
binding in membranes to approximate physiological conditions. This is especially important because PIP2, a component of the cell membrane, plays a
role in G
-mediated activation of GIRKs (52, 53). Nonprenylated
G
mutants do not activate GIRK channels (54, 55), indicating that
G
association with cell membranes may be a prerequisite for
G
binding. We have paid careful attention to detergent
concentrations, because low detergent conditions can potentially expose
hydrophobic patches on GIRKs, producing nonspecific binding. Indeed, we
found it difficult to prevent GIRK and G
aggregation in low
detergent concentrations.
The stoichiometry of the IKACh-G
interaction has been
repeatedly estimated by using the Hill equation to fit the
G
-IKACh dose-response curve. Estimates of the Hill
coefficient for IKACh activation varied from 1.5 (9) to 3 (56, 57) whereas it was ~1 in the study of the direct binding of
purified IKACh and G
proteins (9). Although often
used to infer binding stoichiometry of G
with GIRK subunits, the
Hill coefficient is a measure of cooperativity, not the number of
binding sites. For the Hill coefficient to equal the G
binding
stoichiometry, two criteria need to be met or approximated. The G
molecules must bind the channel simultaneously and G
must bind
with infinite cooperativity (58). In addition, the Hill equation does
not take into account the increasing open probability of the channel
with each ligand molecule bound. Thus, the stoichiometry of G
binding to IKACh is not adequately determined by fits of
the Hill equation to the G
dose-response relations. Even the more
complicated Monod, Wyman, and Changeux (MWC) formula does not properly
describe the subunit gating of the cyclic nucleotide-gated channel
(59). Nevertheless, the cooperativity in IKACh activation (9, 56, 57) and the G
-dependent shifts in its gating modes (60, 61) suggest that GIRK channels have multiple G
binding sites.
Given the inadequacy of available models, a direct biochemical approach
was used to determine GIRK-G
binding stoichiometry. Solutions
containing purified GIRK1 and GIRK4 were treated with the cross-linking
reagent DTSSP in the presence or absence of G
. A 230-kDa band was
observed in the absence of G
, compared with a 390-kDa band when
G
was present. We concluded that the 160-kDa shift was the result
of covalent linkage of multiple 42-kDa G
molecules to the
channel. Next, solubilized recombinant GIRK4 homotetramers were treated
with DTSSP in the presence and absence of G
. A 170-kDa band
formed without G
in contrast to the 320-kDa band in the
G
-containing experiment. The 150-kDa shift in the presence of
G
is most consistent with the chemical cross-linking of four
42-kDa G
molecules to the GIRK4 homotetramers with complete G
binding site saturation and cross-linking. Finally, a variety of membrane-associated GIRK channels were treated with DTSSP and analyzed by SDS-PAGE. In each case, completely cross-linked GIRK tetramers resulted. To verify that there were four G
binding sites in GIRK tetramers, we altered our cross-linking conditions. DTSSP
and G
concentrations were adjusted so that variable numbers of
G
molecules were cross-linked to the GIRK4 homotetramers. Five
adducts, representing zero, one, two, three, and four G
molecules
cross-linked to the channel, were detected. We were unable to
cross-link more than four G
molecules to the channel, even with
G
concentrations two orders of magnitude higher than the
Kd for G
binding to GIRK subunits. We conclude that four G
subunits can bind to a GIRK tetramer.
Currently, the G
binding site on GIRK subunits is thought to
reside primarily on the cytoplasmic C-terminal region shortly after the
second transmembrane domain. But a detailed description of the
GIRK/G
binding site will undoubtedly require direct structural determination. For example, it is not possible to determine with our
experiments whether the G
binding pockets were formed within subunits or between subunits. Short of direct structural determination, in future experiments it may be possible to cross-link G
to GIRKs
during patch clamp recording. Such a technique has proven valuable in
evaluating cyclic nucleotide binding to cyclic nucleotide-gated channels (59).
We would like to thank Matt Kennedy for his
expertise and for providing epitope-tagged GIRK1 and GIRK4, Yiping Chen
for providing technical assistance, Grigory Krapivinsky and Luba
Krapivinsky for purified G
, anti-GIRK1 and anti-GIRK4 antibodies,
Heidi Chial for critical reading of the manuscript and Dr. Eva Neer for
helpful discussions.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M100058200
The abbreviations used are:
PI, phosphatidylinositol;
GIRK, G-protein-gated inwardly rectifying
K+ channel;
DTSSP, dithiobis[sulfosuccinimidylpropionate];
PAGE, polyacrylamide gel
electrophoresis;
Kir, inwardly rectifying K+-selective
channel;
IKACh, native atrial G-protein-gated
K+ channel;
CHO, Chinese hamster ovary cells;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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