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INTRODUCTION |
Since the initial cloning of the Shaker K+ channel in
1987 (1-3), a wealth of K+-selective channels have been
cloned and their electrophysiologic properties characterized. Detailed
structural information on K+-selective channels, however,
has lagged considerably behind. Typically, K+-selective
channels exist at low densities of 1-10 channels/µm2
(4-6), compared with ~ 10,000 channels/µm2 for
the nicotinic receptor (7-9). Because they are membrane proteins, they
require detergents to keep them solubilized. This has made
purification, crystallization, and biochemical characterization difficult. To date, there is no crystal structure or high resolution electron micrographic image of a K+-selective channel.
Of the K+-selective channels, the most structural
information is known about the voltage-gated K+-selective
channels (Kv).1 Kv channels
have been proposed to be tetramers of four identical or highly
homologous subunits, based on a wide variety of methods: toxin binding
studies (10), covalently linked constructs (11), low resolution
electron microscopic imaging (12), and cross-linking studies (13). Each
subunit is thought to consist of 6 transmembrane domains with a loop
contributing to the pore (P-loop) region located between the fifth and
sixth transmembrane domains. Less is known about the inwardly
rectifying K+-selective (Kir) channels. Kir channels are
distantly related to the Kv channels and contain regions equivalent
only to the fifth transmembrane domain, the P-loop, and the sixth
transmembrane domains of the Kv channels. Despite these similarities,
the regions critical for assembly are likely to differ between Kir and
Kv channels . Although the N-terminal domain has been implicated in Kv
assembly (14), the second transmembrane domain and the proximal
C-terminal domains have been implicated in Kir channel assembly (15).
Several groups have suggested that, like Kv channels, Kir channels are
tetramers (16-18).
We are interested in the unique subfamily of Kir channels that are
ligand-gated by direct G
binding (19, 20), the
G-protein-regulated, inwardly rectifying K+ channels
(GIRKs). Using a combination of size exclusion chromatography and
sucrose density gradients, it was originally proposed that GIRK1, in
combination with an unknown subunit, contained three to five subunits
of unknown stoichiometry (21). Later, by studying the biophysical
properties of concatenated subunits, Silverman et al. (22)
suggested that GIRK1 and GIRK4 formed a tetramer in a 1:1
stoichiometry. However, Silverman et al. were unable to
determine a preferred subunit arrangement around the pore and suggested
that more than one arrangement may be viable. Tucker et al.
(23) found that the GIRK1-GIRK4-GIRK1-GIRK4, rather than the
GIRK1-GIRK1-GIRK4-GIRK4 arrangement, produced a higher ratio of
agonist-induced to basal current.
To date, all of the studies on the stoichiometry of Kir channels have
relied upon the formation of multimeric concatemers. Briefly, a single
protein is formed by the translation of a single mRNA encoding
several artificially concatenated subunits. Several combinations of
concatenated subunits are examined, and conclusions are drawn from
their varying properties. These studies have an advantage in that they
define the smallest functional unit, whereas biochemical studies can
define only the smallest physically associating unit. However,
concatemer-based studies assume that: 1) combinations of tandemly
linked subunits that yield the most current are the most representative
of the native channel configuration, 2) tandemly linked subunits do not
coassemble with other tandemly linked subunits, and 3) tandemly linked
subunits are completely translated and are not proteolytically cleaved
between linked subunits. Unfortunately, there are instances in which
each of these assumptions has been proven to be incorrect (18, 22, 24,
25). Silverman et al. (22) were forced to assume that two
trimeric constructs combine to form a functional channel to explain the
high currents produced when only trimeric constructs were expressed. In
addition, these studies have been complicated by the presence of
endogenous oocyte subunits. Despite these drawbacks, in recent years,
the use of tandemly linked subunits has dominated the field of channel stoichiometry and has remained virtually unchecked by other independent methods. Clearly, alternative means must be used to test the validity of this approach.
In this report, we present the first purification of a native mammalian
K+ channel to homogeneity, the first cross-linking study
examining inwardly rectifying K+ channel quaternary
structure and stoichiometry, and the first densitometry study examining
GIRK stoichiometry. Using multiple independent methods, our data
indicate that GIRK channels are tetramers. We hypothesize that native
IKACh is composed of two GIRK1 subunits and two GIRK4
subunits.
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EXPERIMENTAL PROCEDURES |
Purification of Native IKACh and Recombinant
GIRK1-GIRK4 Heteromultimers--
Bovine atrial plasma membranes were
isolated as described (26). Membranes were solubilized in 1.0%
CHAPS-HEDN buffer, pH 7.5 (in mM: 10 HEPES, 1 EDTA, 1 dithiothreitol, and 100 NaCl). The protease inhibitors leupeptin (50 mg/ml, Sigma-Aldrich Inc.), phenylmethylsulfonyl fluoride (100 mg/ml,
Sigma-Aldrich Inc.), aprotinin (1 mg/ml, Sigma-Aldrich Inc.), and
pepstatin (2 mg/ml, Sigma-Aldrich Inc.) were used in all steps of the
purification. Approximately 150 mg of solubilized atrial proteins were
loaded onto a Toyopearl RedTM affinity column. Flow rates were 0.1 ml/min and 1 ml/min during the binding and elution steps, respectively. Bound protein was eluted with the same buffer containing 1 M NaCl. Fractions were assayed for IKACh
subunit content by Western blot. Fractions containing both
IKACh subunits were pooled and dialyzed against 400 mM NaCl containing 1.0% CHAPS-HEDN buffer (pH 7.5). The
equilibrated fractions were concentrated in Centriprep-50TM (Amicon,
Inc., Beverly, MA) concentrators to <2.0 ml and loaded onto a HighLoad
16/60 SuperdexTM (Pharmacia Biotech Inc., Uppsala, Sweden) size
exclusion chromatography column at a flow rate of 0.4 ml/min. Pooled
IKACh fractions were dialyzed against immunoprecipitation (IP) buffer (1% CHAPS, 10 mM HEPES, 100 mM
NaCl, 5 mM EDTA at pH 7.5), loaded onto a wheat germ
agglutinin (WGA, Sigma-Aldrich Inc.) affinity column, and eluted with
0.25 M N-acetylglucosamine. Finally, the eluate
was immunoprecipitated for 150 min at 4 °C with anti-GIRK4 peptide
antibody (anti-CIRN2 was generated against amino acids 19-32; Refs. 19
and 27) and washed three times (1-ml amounts) for 1 min each, three
times (1 ml) for 5 min each, and eluted with 1 mg/ml antigenic peptide
three times (100 µl) for 30 min each at 22 °C.
Plasma membrane proteins containing epitope-tagged GIRK1-AU5 and
GIRK4-AU1 were isolated from COS7 cells as described previously (28).
Samples were precleared for 1 h at 4 °C with 20 µl of Protein
A-Sepharose (Pharmacia Biotech Inc.). The two-step purification consisted of sequential immunoprecipitations in which samples were
first immunoprecipitated with anti-GIRK4 antibodies and were then
immunoprecipitated with anti-GIRK1 antibodies. For the anti-GIRK4 IP,
Protein A-Sepharose was preincubated with 3 µg of anti-CIRN2 (27,
28). After a 30-min preincubation, solubilized proteins from a single
100-mm dish were added and incubated at 4 °C for 150 min. The
Protein A-Sepharose-antibody (Ab)-GIRK complexes were washed five times
(1-ml amounts) with IP buffer. The GIRK heteromultimers were eluted
with 1 mg/ml CIRN2 antigenic peptide for 150 min at 22 °C with three
eluate exchanges. For the anti-GIRK4 IP, the eluate was added to
Protein A-Sepharose and a 1:50 dilution of ascites fluid containing AU5
monoclonal antibodies (BabCO, Berkeley Antibody Co., Richmond, CA) was
incubated at 4 °C for 150 min. The Protein A-Sepharose-Ab-GIRK
complexes were washed five times (1-ml amounts) with IP buffer. GIRK
heteromultimers were eluted with 0.25 mg/ml AU5 antigenic peptide at
22 °C with three 100-µl eluate exchanges for 1 h each.
Chemical Cross-linking--
Protein to be cross-linked was
treated for 1 h on ice with 100 mM dithiothreitol and
dialyzed against 1% CHAPS, 10 mM HEPES, 400 mM
NaCl, pH 8.5 (cross-linking buffer). To block free sulfhydryl groups,
dialyzed aliquots were treated with 25 mM iodoacetamide (Sigma-Aldrich Inc.) for 1 h on ice. Aliquots to be cross-linked via disulfides were not treated with iodoacetamide. Typical reaction volumes were 15 µl for the pure, atrial IKACh and 75 µl
for the recombinant protein. Increasing the reaction volumes >10-fold had no effect on the adducts formed. Approximately 0.1 ng of pure IKACh, 10 µg of crude solubilized atrial protein, or 10 µg of solubilized COS-7 membrane protein was used in each
reaction.
For complete cross-linking, solutions containing 3 mM
dithiobis(sulfosuccinimidylpropionate) (DTSSP, Pierce), 1 mM disuccinimidyl suberate (DSS, Pierce), or 3 mM sulfosuccinimidyl (4-azidophenyldithio)propionate (SSADP, Pierce) were used. Immediately prior to use, cross-linking reagents were prepared as 10 × stock solutions in cross-linking buffer. The water-insoluble DSS was prepared as a 9 × stock
solution in dimethyl sulfoxide (Me2SO). Unless specified
otherwise, the reactions were allowed to proceed for 1 h on ice.
Reactions were terminated for 30 min with 50 mM Tris, pH
7.5, or 15 mM iodoacetamide, when either
H2O2 or iodine was used. The
hetero-bifunctional SSADP was first used like the other reagents, and
then the azide was photoactivated by a 1-min exposure in a CL-1000
ultraviolet cross-linker (UVP, Upland, CA).
For partial cross-linking, dimethyl adipimidate·2HCl (DMA, Pierce)
and dimethyl suberimidate·2HCl (DMS, Pierce) were also used. These
reagents were used at 10 mM final concentrations in 100 mM HEPES containing cross-linking buffer. For DTSSP, SSADP, and iodine, a 10-fold dilution over what was used to completely cross-link the channel generally created a laddering pattern. When
necessary, trichloroacetic acid (Sigma-Aldrich Inc.) precipitation was
used to concentrate the samples prior to SDS-PAGE analysis.
Electrophoresis and Immunoblotting--
Native IKACh
or recombinant GIRK protein was resuspended in Laemmli sample buffer
containing either 50 mM dithiothreitol (reducing conditions) or 25 mM iodoacetamide (non-reducing
conditions) for 30 min at 50 °C. 10% separating and 3% stacking,
3-10% separating and 3% stacking, and precast 2-15% (ISS) gels
were all utilized. Samples were analyzed by fluorography with Amplify
(Amersham Corp.), Gel CodeTM silver staining (Pierce), or by
immunoblotting with anti-GIRK1 antibodies and/or anti-GIRK4 antibodies.
Transfer times for Western blot analysis were extended to >2 h at 15 V
to ensure transfer of the larger cross-linked complexes. When
sequential probing with antibodies was necessary, polyvinylidene
fluoride membranes (Millipore, Bedford, MA) were stripped with 62.5 mM Tris-HCl, 2% SDS, 100 mM 2-mercaptoethanol
for 45 min at 50 °C. When quantitation was required, X-Omat AR film
(Eastman Kodak Co.) was preflashed (SensitizeTM, Amersham Corp.) to
0.15 OD above background and exposed at
70 °C. A GS-700 imaging
densitometer (Bio-Rad) was used to analyze the protein gels and
immunoblots. Molecular weights were calculated using densitometry
profiles from a combination of prestained high molecular weight markers (Bio-Rad) and low and high molecular weight markers (Pharmacia Biotech
Inc.). In a portion of the gels, thyrogloblin (Pharmacia Biotech Inc.)
was added to ensure linearity through at least 330 kDa. When lanes from
several gels were presented together (Figs. 4-6), the molecular weight
markers corresponded to lanes 1 and 2. The
remaining lanes were approximately aligned with the molecular weight
markers. In addition, the molecular weights of all the adducts are
presented in Tables I and II.
Antibody Standardization--
Purified
[35S]methionine-labeled recombinant GIRK1 and/or GIRK4
subunits were purified to homogeneity, as described previously. The
pure GIRK1-GIRK4 heteromultimers were divided into two aliquots, which
were analyzed separately by SDS-PAGE. The first lane was fixed and
exposed to film (Fig. 3B), and the second lane was
transferred to a polyvinylidene fluoride membrane and Western-blotted
along with several lanes of decreasing quantities of solubilized atrial sarcolemma membranes (Fig. 3A). Autoradiography (Fig.
3B) revealed a GIRK1:GIRK4 band intensity ratio of 1.8:1.0
and 1.5:1.0 after a correction for the methionine content of each
protein (see Equation 1). The intensity ratio represents the
stoichiometry of the recombinant protein, but not necessarily the
stoichiometry of the subunits in native atrial IKACh.
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(Eq. 1)
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The aliquot of recombinant GIRK1-GIRK4 that had been
Western-blotted was then analyzed by densitometry. The GIRK1:GIRK4 band intensity ratio was 8:1 and will be referred to as the Ab stoichiometry (apparent stoichiometry as detected by antibody). The Ab stoichiometry is measured from the ratio of the intensity of Western blot bands and
is a product of the number of moles of each individual subunit and the
number of Abs bound to each epitope.
Dividing the recombinant stoichiometry by the Ab stoichiometry yields a
useful term, which we will refer to as the Ab standardization factor
(Equation 2).
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(Eq. 2)
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In our case, the Ab standardization factor was 0.19 (0.19 = 1.5/8). The Ab standardization factor multiplied by the GIRK1:GIRK4 band intensity ratio of the Western-blotted native channel yields the
true stoichiometry of the native channel (Equation 3).
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(Eq. 3)
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In our case, the true stoichiometry of native IKACh
was ~1:1 (0.19 × 6 = 1.1).
The above procedure assumes that there is a direct linear relationship
between the Western blot intensity and the total amount of protein on
the blot and that this relationship is maintained for both antibodies
over the range of the protein concentrations to be tested. We found
that our antibodies satisfied this criteria; the intensity of the lanes
from Western-blotted atrial sarcolemma membrane varied in a direct and
linear manner with the amount of protein.
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RESULTS |
Purification of Native IKACh and Recombinant
GIRK1-GIRK4 Heteromultimers--
We have purified native bovine
IKACh and recombinant GIRK1-GIRK4 heteromultimeric channels
to near homogeneity. Both purification procedures were specifically
designed to purify only heteromultimeric channels composed of GIRK1 and
GIRK4 subunits (Fig. 1). Potential homomultimeric channels, or dissociated monomers, were not purified with these procedures.

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Fig. 1.
Purification schemes designed to select for
heteromultimeric channels. A, purification of native
IKACh. A combination of WGA (wheat germ agglutinin)
chromatography and immunoprecipitation with anti-GIRK4 antibodies was
used to purify native heteromultimeric channels. B,
purification of recombinant GIRK1-GIRK4 heteromultimers. Sequential
immunoprecipitation with anti-GIRK4 and anti-GIRK1-AU5 tag antibodies
was used to purify recombinant heteromultimeric channels. A tetrameric
complex is assumed for the purpose of the diagram. Question
mark (?), either GIRK1 or GIRK4; branched tails on
circles, glycosylated.
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Native IKACh was purified from isolated bovine atrial
plasma membranes (Fig. 1A). The membranes were solubilized
in 1% CHAPS and subjected to the following purification steps: 1)
Toyopearl RedTM affinity chromatography, 2) size exclusion
chromatography, 3) WGA affinity chromatography, and 4) IP with
anti-GIRK4 antibodies followed by elution with antigenic peptide. WGA
affinity chromatography was specific for GIRK1, because GIRK4 is not
glycosylated (39). Thus, the combination of WGA affinity chromatography
and immunoprecipitation with anti-GIRK4 antibodies ensured that no
homomultimeric channels or dissociated monomers were purified. The
final product, purified to greater than 95% homogeneity, was native
bovine atrial IKACh. Aliquots of purified native
IKACh were analyzed by SDS-PAGE and silver-stained (Fig. 2,
lane 1) or immunoblotted with anti-GIRK4 antibodies (Fig. 2,
lane 2) and then stripped and reimmunoblotted with
anti-GIRK1 antibodies (Fig. 2, lane 3). The predominant
bands in the silver-stained lane were also recognized by anti-GIRK1 and
anti-GIRK4 antibodies when Western-blotted. The bands correspond to
GIRK4 (48 kDa), GIRK1 (54 kDa), and glycosylated GIRK1 (56-76 kDa).
Interestingly, the WGA affinity chromatography enhanced the proportion
of glycosylated to unglycosylated GIRK1 when compared with unpurified
channels, but did not completely eliminate unglycosylated GIRK1
(Fig. 2A, 54-kDa band).
Presumably, some proportion of the native IKACh
heteromultimers contain both glycosylated and unglycosylated GIRK1
subunits.

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Fig. 2.
Purified native IKACh and
recombinant GIRK1-GIRK4 heteromultimers. Purified native
IKACh and recombinant GIRK1-GIRK4 heteromultimers were
analyzed by 10% SDS-PAGE. A, purified native IKACh was silver-stained or immunoblotted with anti-GIRK4
antibodies ( GIRK4) or anti-GIRK1 antibodies
( GIRK1). Presumably, some channel complexes contained at
least one glycosylated (56-76 kDa) and one unglycosylated GIRK1 (54 kDa) subunit, which allowed the unglycosylated GIRK1 subunit to be
co-purified. Densitometry analysis of lane 1 was consistent
with a 1:1 GIRK1:GIRK4 stoichiometry. B, purified recombinant GIRK1-GIRK4 heteromultimers were immunoblotted
simultaneously with anti-GIRK1 and anti-GIRK4 antibodies
( GRIK1/4, lane 1) or autoradiographed
(lane 2). The majority of bands labeled by silver staining
or [35S]methionine ([35S]Met)
were also recognized by antibody. gly,
glycosylated.
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Recombinant GIRK1-GIRK4 heteromultimeric channels were purified from
transiently transfected COS7 cells using sequential anti-GIRK1 and
anti-GIRK4 immunoprecipitations (see Fig. 1B and
"Experimental Procedures"). The sequential immunoprecipitations
assured that only heteromultimeric channels were purified. The purified
protein was analyzed by SDS-PAGE, followed by either Western blotting or autoradiography (Fig. 2B). All of the protein bands that
appeared on the autoradiogram were also recognized by anti-GIRK1 or
anti-GIRK4 antibodies.
Densitometry of Silver-stained Native IKACh and
[35S]Methionine-labeled Recombinant Proteins Suggests a
1:1 GIRK1 to GIRK4 Subunit Stoichiometry for Native
IKACh--
Two independent methods based on densitometry
were used to examine GIRK1:GIRK4 stoichiometry. In the first method,
silver-stained SDS-PAGE gels of purified native IKACh were
analyzed by densitometry (Fig. 2A, lane 1). The
ratio of band intensities for GIRK1:GIRK4 was 1.2:1. When this ratio
was corrected by multiplying it by the predicted unglycosylated
molecular weight of GIRK4(47 kDa)-GIRK1(56 kDa), a molar ratio of 1:1
(n = 3) for GIRK1:GIRK4 was obtained. The advantage of
this procedure is that it examines GIRK1:GIRK4 stoichiometry as it
exists in native atrial tissue. GIRK1 and GIRK4 share 57% overall
amino acid identity, and the silver-stained bands representing GIRK1
and GIRK4 proteins varied by less than 3-fold in intensity. These GIRK1
and GIRK4 similarities minimize the potential inaccuracies of protein
quantification by silver staining.
The second independent method used to examine GIRK1:GIRK4 stoichiometry
was based on the [35S]methionine labeling of purified
recombinant COS7 GIRK1 and GIRK4 heteromultimers. Because the number of
counts emitted by a radiolabeled subunit is directly proportional to
its methionine content, this method more accurately quantifies GIRK1
and GIRK4. Ideally, we could determine the native atrial GIRK1:GIRK4
stoichiometry by comparing 35S-labeled GIRK1 and GIRK4
(Fig. 2B). However, the stoichiometry may vary with the
amount of RNA injected into oocytes (29, 30). If the stoichiometry
varied in COS-7 cells, as well, this approach would be inadequate for
determining native stoichiometry. Indeed, we found that the GIRK1:GIRK4
stoichiometry of heteromultimeric channels did vary directly with the
ratio of GIRK1:GIRK4 DNA used to transfect the COS-7 cells (data not
shown). We were unable to force the GIRK1:GIRK4 stoichiometry beyond
1:3 or 3:1 by varying the ratio of GIRK1:GIRK4 DNA transfected by
30-fold, supporting the conclusion that recombinant GIRK1 and GIRK4
subunits form tetramers.
Because the recombinant system does not necessarily reflect native
stoichiometry, we developed a hybrid approach that combined the
accurate qauntitation achievable with [35S]methionine
labeling of recombinant proteins with the ability of our antibodies to
detect native protein. This method involved standardizing the relative
blotting sensitivities of our anti-GIRK1 and anti-GIRK4 antibodies with
a known ratio of labeled recombinant protein. The standardized
antibodies were then used to probe native atrial protein and the true
native stoichiometry was computed as ~1:1 (see "Experimental
Procedures" and Fig. 3). Previously, Huang et al. (31) showed that the ratio of two antigens
could be estimated with confidence using a similar antibody-blotting method.

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Fig. 3.
Immunoblotting native IKACh with
standardized antibodies yields 1:1 GIRK1:GIRK4 stoichiometry.
[35S]Methionine-labeled recombinant GIRK1 and GIRK4 were
used to standardize anti-GIRK1 and anti-GIRK4 antibodies. The
standardized antibodies were then used to immunoblot native
IKACh. A, lane 1,
[35S]methionine-labeled, recombinant GIRK1 and GIRK4;
lanes 2-5, 2.0, 1.0, 0.5, 0.25 µg, respectively, of
solubilized atrial membrane protein. All lanes were analyzed by 10%
SDS-PAGE and immunoblotted with anti-GIRK1 and anti-GIRK4 antibodies.
B, [35S]methionine-labeled recombinant GIRK1
and GIRK4 (rGIRK1 and rGIRK4) were analyzed by
10% SDS-PAGE followed by autoradiography.
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Complete Cross-linking of Native Purified
IKACh--
We extensively cross-linked native bovine
IKACh (Fig. 4), recombinant
GIRK1 homomultimers (Fig. 5), and
recombinant GIRK4 homomultimers (Fig. 5) using a wide variety of
cross-linking reagents. First, purified native IKACh was
treated with the highly reactive, amine-specific,
N-hydroxysuccinimide (NHS) ester, DTSSP. This reaction
produced a multimeric protein detected as a single 234 ± 7-kDa
band (n = 8). This cross-linked product was recognized by both anti-GIRK1 (Fig. 4, lane 4) and anti-GIRK4 (Fig. 4,
lane 3) antibodies, indicating that both GIRK1 and GIRK4
were cross-linked. The lipid-soluble NHS ester, disuccinimidyl suberate
(DSS), yielded a nearly identical 235-kDa band (Fig. 4, lane
5). Finally, the entire complex was cross-linked through simple
oxidation with iodine (Fig. 4, lane 6), and similar
results were obtained.

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Fig. 4.
Complete cross-linking of native
IKACh yields products that are most consistent with
tetrameric channel formation. Native IKACh was treated
with a wide variety of cross-linking reagents. Cross-linked products
were then analyzed by SDS-PAGE and immunoblotted. Lane 1, no
cross-link control immunoblotted with anti-GIRK1 and anti-GIRK4
antibodies ( GIRK1 and GIRK4). Lane
2, solubilized atrial membrane proteins treated with 3 mM SSADP followed by photolysis for 1 min and immunoblotted
with anti-GIRK1 antibodies. Lane 3, pure IKACh
treated for 1 h with 3 mM DTSSP and immunoblotted with
anti-GIRK1 antibodies. Lane 4, lane 3 stripped
and reimmunoblotted with anti-GIRK4 antibodies. Lane 5, pure
IKACh treated for 1 h with 1 mM DSS and
immunoblotted with anti-GIRK4 antibodies. Lane 6, pure
IKACh treated for 1 h with 50% saturated iodine and
immunoblotted with anti-GIRK4 antibodies. Molecular weight markers were
run with lanes 1 and 2; lanes 3-6 were derived from separate gels and aligned based on corresponding molecular weight standards. See Table I for a summary of averaged molecular weights.
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Fig. 5.
Native IKACh heteromultimers and
recombinant GIRK homomultimers form similar oligomeric structures.
Despite the inability of GIRK1 subunits to produce functional channels
alone, GIRK1 subunits form oligomeric structures similar to native
IKACh heteromultimers and GIRK4 homomultimers.
A, native IKACh and recombinant GIRK1 or GIRK4
homomultimers were chemically cross-linked. Cross-linked products were
then analyzed by SDS-PAGE and immunoblotted. The molecular weights of
the cross-linked native IKACh, recombinant GIRK1, and
recombinant GIRK4 homomultimers were all consistent with tetrameric
complex formation. Lane 1, no cross-link control immunoblotted with anti-GIRK1( GIRK1) and
anti-GIRK4( GIRK4) antibodies. Lane 2,
solubilized atrial membrane proteins treated with 3 mM SSADP followed by photolysis for 1 min and immunoblotted with anti-GIRK1 antibodies. Lane 3, recombinant GIRK1
homomultimers treated for 1 h with 3 mM DTSSP and
immunoblotted with anti-GIRK1 antibodies. Lane 4,
recombinant GIRK4 homomultimers treated for 1 h with 3 mM DTSSP and immunoblotted with anti-GIRK4 antibodies. Molecular weight markers were run with lanes 1 and
2; lanes 3 and 4 were derived from
separate gels and aligned based on corresponding molecular weight
standards. See Table I for a summary of averaged molecular weights.
B, native IKACh and recombinant GIRK1 elute at
similar volumes during size-exclusion chromatography. The portion of
the channel that eluted with the void volume
(Vo) varied between trials. No attempt was made
to estimate the molecular weight of the channel due to the
complications caused by detergent binding.
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To address the potential concern that the native complex is composed of
an integral multiple of the ~235-kDa complex, or that associating
proteins were lost, native IKACh protein was treated with
an even more highly reactive cross-linking agent. The
heterobifunctional NHS ester/aryl azide SSADP was used to cross-link
native IKACh immediately after solubilization and prior to
any further purification to prevent potential subunit degradation or
dissociation. SSADP cross-linked protein (like DTSSP, DSS, and iodine)
was detected as a single 224 ± 3-kDa (n = 4) band
(Fig. 4, lane 2). Monomers were detected only after extended
exposure to film, if at all (data not shown). This indicates that the
entire GIRK1-GIRK4 complex was intact after purification because
dissociated monomers would have remained at the bottom of the gel.
Cross-linking of recombinant GIRK1 (Fig. 5A, lane
3) and GIRK4 (Fig. 5A, lane 4) homomultimers also produced a single unique complex of 216 ± 22 kDa
(n = 4) and 212 ± 13 kDa (n = 3),
respectively. If significant amounts of interchannel cross-linking
rather than intrachannel cross-linking had occurred, a smear would have
appeared at the top of the gel. A tetramer made up of equal numbers of
GIRK1 subunits (approximately 65 kDa with glycosylation) and GIRK4
subunits (approximately 48 kDa) would have had a molecular weight of
~226 kDa. Thus, the total molecular weight of the native cross-linked
IKACh complex, ~234 kDa, is consistent with a tetramer.
Similarly, the total molecular weights of completely cross-linked
homomultimeric GIRK1 ~216-kDa channel (222 kDa, predicted) and GIRK4
~211-kDa channel (188 kDa, predicted) are consistent with a tetramer.
Finally, we found that recombinant GIRK1 homomultimers eluted to a
position similar to that for native IKACh during size
exclusion chromatography (Fig. 5B). The size exclusion
chromatography and complete cross-linking experiments support a similar
oligomeric structure for GIRK1 homomultimers and the native
IKACh heteromultimer. A summary of the various cross-linking reactions is given in Table
I.
Partial Chemical Cross-linking of Native IKACh Reveals
Monomeric, Dimeric, Trimeric, and Tetrameric Complexes--
Another
approach to testing the tetrameric channel hypothesis involves analysis
of partially cross-linked native IKACh. Previously, it was
shown that the molecular weight of partially cross-linked proteins
increases in a linear fashion with the number of cross-linked subunits
(32). In this experiment, native IKACh was cross-linked with DMA, an imidoester, or with DTSSP, the more reactive NHS ester.
The electrophoretic pattern produced following SDS-PAGE and Western
blotting is shown in Fig. 6. The blot was first probed with anti-GIRK4
antibodies (Fig. 6A,
lanes 1-3) and then completely stripped and reprobed with
anti-GIRK1 antibodies (Fig. 6A, lanes 4-6).
Partial cross-linking of native IKACh produced a laddered pattern consisting of four main adducts (Fig. 6A). The four
adducts represent (from bottom to top) monomeric (41-61 kDa), dimeric (94-138 kDa), trimeric (~185 kDa), and tetrameric (~231 kDa) forms of the channel (Fig. 6A). The adducts formed in a manner in
which the proportion of higher molecular weight adducts increased with increasing cross-linking times (Fig. 6, lane 1 versus lane
2) or cross-linking agent concentrations (data not shown). By
densitometric scanning of Western blots (Fig. 6A,
lanes 1 and 4), GIRK1 and GIRK4 antigenicity
profiles were developed (Fig. 6B). Cross-linking ladders
were also created when native IKACh was treated with DMS, iodine, or DSS (Fig. 6, C, D, and
inset to D). In addition, partial cross-linking
of recombinant homomultimeric GIRK4 and GIRK1 channels yielded a
laddered pattern which was most consistent with homotetrameric proteins. The mean molecular weights of the various adducts produced by
cross-linking of native atrial IKACh and recombinant
homomultimeric channels are summarized in Table
II.

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Fig. 6.
Partial cross-linking of pure, native
IKACh is most consistent with a tetramer composed of two
GIRK1 subunits and two GIRK4 subunits. Partial cross-linking
produces adducts that represent monomers, dimers, trimers, and
tetramers. A, pure, native IKACh was treated
with 10 mM DMA for 15 min (lanes 1 and
4), 2 h (lanes 2 and 5), or DTSSP
for 1 h (lanes 3 and 6). The products were analyzed by 3-10% SDS-PAGE and immunoblotted. Lanes 1-3
were immunoblotted with anti-GIRK4 antibodies; lanes 4-6
correspond to lanes 1-3 when stripped and immunoblotted
with anti-GIRK1 antibodies. B, anti-GIRK1
( GIRK1) and anti-GIRK4 ( GIRK4) antigenicity
profiles were created by densitometry scanning of lanes 2 and 4 of A. Likewise, profiles created when
native IKACh was treated with either iodine (C),
DMS (D), or DSS (D, inset) are shown.
gly, glycosylated.
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Table II
Partial cross-linking of GIRK channels
1, GIRK1; 4, GIRK4. Molecular weights are given as a mean in kDa ± standard deviation (n, number of trials).
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An Examination of Specific Dimer, Trimer and Tetramer Adducts
Confirms That Native Atrial IKACh Is a Heterotetramer
Composed of Two GIRK1 and Two GIRK4 Subunits--
Examination of
dimers formed by partial cross-linking of purified native atrial
ICh protein reveals GIRK4-GIRK4, GIRK1-GIRK4, and
GIRK1-GIRK1 adducts (Fig. 6). As expected, the relative proportions of
the specific dimeric adducts that formed depended on the side chain
specificity, cross-linking span, and lipid solubility of the
cross-linking agent. The formation of the GIRK1-GIRK1 and GIRK4-GIRK4
adducts demonstrated that native atrial IKACh
heterotetramers are composed of two GIRK1 and two GIRK4 subunits and
corroborates the previous densitometry experiments. The trimeric peak
in Fig. 6C is composed of GIRK1-GIRK1-GIRK4 and
GIRK4-GIRK4-GIRK1 adducts. As expected, the trimeric peak is
broad and the GIRK4 peak antigenicity is shifted toward the lower
molecular weights (~179 kDa), whereas the GIRK1 peak antigenicity is
shifted toward the higher molecular weights (~188 kDa). On no
occasion did such an antigenicity shift occur in the
tetrameric adduct. The simplest interpretation of this
pattern is that the native IKACh tetramer is composed of a
single population of channels with two GIRK1 and two GIRK4
subunits.
 |
DISCUSSION |
We report the purification of a native mammalian K+
channel to near homogeneity and provide direct biochemical evidence for IKACh channel stoichiometry and quaternary structure. Using
numerous independent methods, we have shown that GIRK proteins form
tetramers and that native IKACh is most likely a tetramer
composed of two GIRK1 subunits and two GIRK4 subunits.
After purification of native IKACh to greater than 95%
homogeneity, we found that the channel was comprised of GIRK4 (48 kDa), GIRK1 (54 kDa), and glycosylated GIRK1 (56-76 kDa) subunits. The complex tightly bound WGA during purification, indicating that it
contained terminal sialic acid residues. The purified product cross-linked into a single high molecular weight complex, indicating that the purified channel was an intact tetramer. The high degree of
native channel protein purity was the key to our experiments because it
eliminated potential nonspecific cross-linking between native
IKACh and other unrelated membrane proteins. We cannot rule
out the possibility that other populations of GIRK1-GIRK4 heteromultimers with alternate stoichiometries did not copurify. However, immunodepletion experiments illustrate that greater than 90%
of GIRK4 is associated with GIRK1 (27) and that greater than 90% of
GIRK1 is associated with GIRK4 (data not shown). These immunodepletion
experiments verify the lack of significant quantities of native
homomultimeric complexes, if they exist at all.
Chemical cross-linking has been widely used in the nearest neighbor
analysis of membrane proteins and to study subunit organization (see
Refs. 32-34 for review). The total number of subunits in both the
glycine receptor (25) and Shaker channels (13) have been estimated by cross-linking approaches. Here, we have demonstrated that
GIRK channels are tetrameric complexes by using two chemical cross-linking approaches. In the first approach, we cross-linked purified native IKACh heteromultimers, recombinant GIRK1
homomultimers, and recombinant GIRK4 homomultimers. Four reagents with
different side chain specificities, lipid solubilities, and
cross-linking spans all produced a single unique adduct, strongly
indicating that the channels were purified in an intact state and then
completely cross-linked. Native IKACh heteromultimers,
recombinant GIRK1 homomultimers, and recombinant GIRK4 homomultimers
formed complexes of ~234, 216, and 211 kDa, respectively, consistent
with predicted molecular weights of ~226, 222, and 188 kDa,
respectively. The use of cross-linking agents and iodoacetamide
potentially complicates the interpretation of our results, because both
of these reagents can covalently bind the protein and therefore might
increase its apparent molecular weight. These agents may also alter the
protein's mobility characteristics by changing its charge,
hydrodynamic properties, or SDS binding. Nonetheless, others have shown
that the molecular weight of cross-linked complexes determined by
SDS-PAGE closely approximated the true molecular weight of
heteromultimeric proteins (25, 32, 35, 36). On balance, the internal
consistency of our results suggests that native IKACh
components were purified to homogeneity and cross-linked into one
complete complex. To verify that the products resulting from complete
cross-linking were tetramers, we partially cross-linked native atrial
IKACh, homomultimeric GIRK4, and homomultimeric GIRK1
channels. Partial cross-linking formed four adducts representing
monomers, dimers, trimers, and tetramers. The molecular weight of the
adducts increased with the total number of subunits cross-linked in a
linear fashion, again supporting the conclusion that native atrial
IKACh heteromultimers, recombinant GIRK1 homomultimers, and
recombinant GIRK4 homomultimers all formed tetrameric complexes.
Three species of dimers, GIRK1-GIRK1, GIRK1-GIRK4, and GIRK4-GIRK4,
were detected when native IKACh was partially cross-linked, based upon interpretation of molecular weights and Western blotting. The formation of GIRK1-GIRK1 and GIRK4-GIRK4 cross-linked subunits within the native IKACh tetramer indicated that the
tetramer is composed of two GIRK1 subunits and two GIRK4 subunits in
~1:1 stoichiometry. The 1:1 stoichiometry was supported by two
additional experimental approaches. First, silver-stained gels of
purified IKACh yielded a 1:1 GIRK1:GIRK4 staining intensity
ratio after correction for their respective molecular weights. Both the
purification scheme and cross-linking experiments assured that only
complete tetrameric heteromultimers were examined by densitometry.
Second, immunoblotting of native IKACh was consistent with
1:1 stoichiometry provided that the blotting antibodies were first
standardized against a known ratio of recombinant GIRK1 and GIRK4
proteins. Thus, three different experimental methods support the
conclusion that GIRK1:GIRK4 stoichiometry is 1:1, and rule out a fixed
3:1 or 1:3 GIRK1:GIRK4 stoichiometry.
Experimental results similar to those shown here might have resulted
from a random assembly of GIRK1 and GIRK4 subunits. This is an
intriguing possibility, considering that in our heterologous COS7
expression system, the stoichiometry of GIRK1:GIRK4 varied directly
with the ratio of DNA transfected, and would provide yet another way to
contribute to K+ channel diversity. However, this
interpretation would require that the 3:1 and 1:3 GIRK1:GIRK4 pools
were of equal size to be compatible with the 1:1 stoichiometry
determined by densitometry. Furthermore, large pools of complexes with
1:3 and 3:1 stoichiometries would not be consistent with the observed
nearly identical anti-GIRK1 and anti-GIRK4 tetrameric adduct profiles.
Thus, we currently favor the simplest interpretation of the data, which
is a fixed 2:2 GIRK1:GIRK4 stoichiometry rather than a random
association model.
Previous work has demonstrated that GIRK4 in expression systems may
form homomultimeric ion channels (27, 37, 39), whereas putative GIRK1
homomultimers are not functional (37-39 ). Moreover, GIRK1, by itself,
does not localize to the membrane (28). One possible explanation for
these findings is that GIRK1 is unable to assemble with itself to form
a tetramer, and is instead shuttled into a degradative pathway as
monomers or aggregates upon translation. It thus appears that GIRK1
must form heteromultimers with another GIRK family member to function.
Wischmeyer et al. (30) suggest that GIRK1 homomultimers may
not exist in vivo due to the spatial conflict of bulky
phenylalanines in the pore structure. In this study, we show that
recombinant GIRK1 subunits can form homotetrameric complexes, although
all evidence to date suggests they are not functional.
In summary, the experiments presented here demonstrate that GIRK
channels form tetramers and that IKACh is most likely made up of two GIRK1 subunits and two GIRK4 subunits. Although we cannot rule out the possibility that there exist multiple pools of channels with varying stoichiometries, the consistency of channel conductances and kinetics from single-channel recordings in numerous species make
this unlikely. This study lays the foundation for future biochemical
studies on G
binding stoichiometry, and determination
of K+ channel subunit arrangement around the pore.
We thank Matt Kennedy and Kevin Wickman for
critically reading the manuscript, Matt Kennedy for expertise and for
providing epitope-tagged GIRK1 and GIRK4, and Yiping Chen for providing technical assistance.