(Received for publication, September 22, 1995; and in revised form, October 19, 1995)
From the
The cardiac G protein-gated K channel,
I
, is activated by application of purified and
recombinant
and
subunits (G
) of heterotrimeric G
proteins to excised inside-out patches from atrial membranes
(Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E., and Clapham, D.
E.(1987) Nature 325, 321-326; Wickman, K.,
[Medline]
Iniguez-Lluhi, J., Davenport, P., Taussig, R. A., Krapivinsky, G. B.,
Linder, M. E., Gilman, A., and Clapham, D. E. (1994) Nature 368, 255-257). Cardiac I
[Medline]
is composed of two
inward rectifier K
channel subunits, GIRK1 and CIR
(Krapivinsky, G., Gordon, E., Wickman, K., Velimirovic, B.,
Krapivinsky, L., and Clapham, D. E.(1995) Nature 374,
135-141). We show that G
directly binds to
immunoprecipitated cardiac I
as well as to recombinant
CIR and GIRK1 subunits, with dissociation constants (K
) of 55, 50, and 125 nM,
respectively. In each case, binding appeared specific as judged by
competition of unlabeled G
with radiolabeled G
and
inhibition of binding by antigenic peptide or G
-GDP, but not
G
-GTP
S (guanosine 5`-3-O-(thio)triphosphate). In
contrast, G
(GTP
S- or GDP-bound) did not bind to the native
channel. We conclude that G
binds directly and specifically
to I
via interactions with both CIR and GIRK1 subunits
to gate the channel.
Acetylcholine (ACh) ()secreted from the vagus nerve
binds cardiac muscarinic receptors, initiating a sequence of events
leading to slowing of the heart rate. I
, an inwardly
rectifying, potassium-selective channel stimulated by the
and
subunit (G
) of pertussis toxin-sensitive heterotrimeric
G proteins, mediates part of this process by hyperpolarizing pacemaker
cells in sinoatrial and atrioventricular nodes of the
heart(4, 5, 6, 7) . All evidence
indicates that the critical components involved in I
activation are confined to the membrane. However, it is unclear
whether G
binds directly to I
to elicit the
stimulatory effect or acts via an unknown intermediate
step(s)(4, 5) .
Cardiac I is a
heteromultimer of two homologous inward rectifier K
channel subunits, GIRK1 (8, 9) and
CIR(3) . Recent evidence also suggests that a similar complex
comprised of GIRK1 and GIRK2(10) , a structural and functional
CIR homolog, forms a neuronal G protein-gated K
channel(11) . We tested whether G
binds directly to
cardiac I
and, if so, to which subunit(s). By
immunoprecipitation with subunit-specific antibodies, we were able to
effectively purify whole cardiac I
channel and
individual recombinant subunits and study binding of
I-labeled G
to these immunoprecipitated
species. Here we show that G
binds directly and specifically
to the whole channel and to each I
subunit.
Electrophysiological experiments with atrial myocytes were
performed as described(2) . The pipette and bath solutions were
identical (in mM): 140 K, 144
Cl
, 5 EGTA, 2 Mg
, 10 HEPES, pH 7.2.
Holding potential was -80 mV. Spaces in the upper trace (see Fig. 1A) represent only the time required to
add the indicated substance to the bath. The lower, expanded trace in Fig. 1A was filtered (8-pole
Bessel) at 2.5 kHz. The concentration-response analysis in Fig. 1B was performed as described(2) . The
data were fit to the sigmoid function f(x) = (a - d)/(1 + (x/c)
) + d using the Marquardt-Levenberg least squares curve-fitting
algorithm (a and d represent the asymptotic maximum
and minimum, respectively; c is K
, the
value of x at the inflection point; b is the Hill
coefficient).
Figure 1:
G activates I
in inside-out patches from rat atrial myocytes. A, 10
nM bovine brain G
consistently activated
I
. Subsequent addition of 30-100 nM bovine brain G
-GDP completely inhibited I
activity elicited by G
. Channel activity was restored
by the addition of excess G
. B, dependence of
I
activity (relative Np
)
on G
concentration. The cumulative K
and the Hill coefficient determined by averaging data obtained
for bovine brain G
and five recombinant G
complexes
(includes data reported in (2) ) were 5 nM and 1.5,
respectively. The table inset shows the relevant parameters
determined for each type of G
preparation. Np
values are normalized to
subsequent GTP
S stimulation (Np
= 1).
G-proteins were isolated from bovine brain, separated
into G and G
subunits as described(12) , and
additionally purified by affinity chromatography over immobilized
G
(13) or G
(14) . Bovine atrial plasma
membranes were isolated as described(15) . Membranes were
solubilized in 1.0% CHAPS-HEDN buffer (in mM: 10 HEPES, 1
EDTA, 1 dithiothreitol, and 100 NaCl) containing protease inhibitors.
Two different antipeptide affinity-purified antibodies were used for
immunoprecipitation experiments: anti-CIR (aCIRN2, amino acids
19-32, 0.5 µg/assay) and anti-GIRK1 (aCsh, amino acids
356-501, 0.3 µg/assay of atrial membrane protein and 0.7
µg/assay of Sf9 membrane protein). aCIRN2 did not immunoprecipitate in vitro translated GIRK1, and aCsh did not immunoprecipitate in vitro translated CIR (3) . (
)Proteins
were immunoprecipitated for 1.5 h at 4 °C with corresponding
antibody and PrA FF-Sepharose (Pharmacia Biotech Inc.).
Immunoprecipitates were washed four times in the same buffer, followed
by two washes with 0.1% CHAPS-HEDN. Anti-G
antibody was purchased
from Calbiochem.
For radiolabeling of G protein subunits, 20 µg
of purified protein was labeled with I using 250 µCi
of
I-Bolton-Hunter reagent (DuPont-NEN) yielding
1
mol of
I/3 mol of G-protein subunit.
I-Bolton-Hunter reagent, at this stoichiometry, does not
prevent formation of a functional heterotrimer by labeled subunits.
Both labeled G
and G
were able to bind their unlabeled
immobilized counterparts, and incubation with
AlF
led to their dissociation
(data not shown). For each binding assay, immunoprecipitates were
obtained from 50 µg of atrial membrane protein and 50 or 500 µg
of Sf9 membrane protein containing rCIR or rGIRK1, respectively.
Immunoprecipitated proteins were incubated with 1.25 nM
I-G
(
10
cpm) and
unlabeled competitors in 0.1% CHAPS-HEDN and rotated for 15 min at room
temperature (75 µl, total volume). Subsequently, the Immunobeads
were washed four times by centrifugation, each time using 0.5 ml of the
same ice-cold buffer. Total washing time was 7 min. In control
experiments, the amount of bound G
did not increase after 15
min at room temperature, and t
for dissociation of
bound G
was
90 min at 4 °C. Bound G
was
counted using a
counter. Data were fit to a competition equation
with a single binding site(17) .
Recombinant baculoviruses containing the 5`-untranslated region and coding region of GIRK1 and the coding region of CIR (3) were produced using the non-fusion baculovirus transfer vector pBlueBac III. The viruses were generated, isolated, and amplified as described (MaxBac, Invitrogen). Five days after infection, cells were harvested and homogenized in a hypotonic buffer. Membranes were then collected, solubilized, and immunoprecipitated as described for atrial membranes.
The functional interaction between G and I
has been well studied in inside-out membrane patches from atrial
myocytes. Bovine brain G
reproducibly activates
I
, and as expected for a G
-dependent process,
channel activity is inhibited by excess G
-GDP (Fig. 1A; see also Refs. 1, 2, 6, and 7). Inhibition by
G
-GDP is overcome by excess G
(Fig. 1A;
see also (2) ). Because there was no statistically significant
difference between the potency of bovine brain G
and the
potencies of all G
recombinant subunits tested previously
(except transducin G
1
1; (2) ), these data were
averaged to generate the cumulative concentration-response relation
shown in Fig. 1B. The resultant K
and the Hill coefficient, as determined by the best fit of the
cumulative data, were 5 nM and 1.5, respectively.
This type
of functional study cannot address whether there is a direct
interaction between G and I
. To examine this
issue, we studied G
binding to the channel. An anti-peptide
antibody (aCIRN2) directed against a unique amino-terminal domain of
the CIR subunit immunoprecipitated CIR and coimmunoprecipitated GIRK1
from bovine atrial membranes.
Endogenous cardiac
G
bound the native channel (Fig. 2A).
Significantly, native cardiac G
associated with the
aCIRN2-immunoprecipitated channel complex only when atrial sarcolemmal
membranes were treated with GTP
S to activate endogenous G
proteins. This suggests that prior to activation, G protein
heterotrimers do not complex with I
. Although the
association of native cardiac G
with the channel was clear,
the signal was inadequate for accurate quantitation of binding.
Figure 2:
G binds to native atrial
I
. A, G
binds to I
only after dissociation of endogenous heterotrimeric G proteins
by GTP
S. Bovine atrial plasma membranes (1 mg) were treated with
100 µM GTP
S, solubilized in 1.0% CHAPS-HEDN, and
immunoprecipitated by aCIRN2. Immunoprecipitated proteins were Western
blotted with an anti-G
antibody. B,
I-G
binds to cardiac I
immunoprecipitated by the anti-CIR antibody, aCIRN2. Binding of
1.25 nM
I-G
was inhibited by 200
µM CIRN2 antigenic (Ag) peptide, 1.3 µM unlabeled G
, and 125 nM G
-GDP. In
contrast, virtually no inhibition was observed with 125 nM G
-GTP
S. For this and Fig. 3, A and B, 100 on the y axis refers to full binding of
I-G
in the absence of competing proteins. C, competition of unlabeled G
and
I-G
for binding sites on I
was
used to evaluate the equilibrium binding constant. Binding of
I-G
to aCIRN2-precipitated I
in
0.1% CHAPS (
) or 0.1% Lubrol PX (
) and binding of
I-G
to aCsh-precipitated I
in
0.1% CHAPS (
) is shown.. All data points represent the average of
three separate experiments. The data were fit well to a model
consisting of a single type of binding
site.
Figure 3:
G binding to recombinant CIR and
GIRK1 subunits. A and B demonstrate the specificity
of G
binding to recombinant CIR (rCIR) or to
recombinant GIRK1 (rGIRK1). Recombinant CIR and recombinant
GIRK1 were immunoprecipitated with aCIRN2 and aCsh, respectively.
Conditions were identical to those described in the legend to Fig. 2. Nonspecific binding for aCsh immunoprecipitates was
determined using 500 µg of wild type Sf9 cell membranes. C, binding affinity of G
to recombinant CIR and
recombinant GIRK1. Nonspecific binding was subtracted, and each point was normalized to maximal binding in absence of
competitor.
To
quantify binding of G to the channel, we measured the binding
of
I-labeled, purified bovine brain G
to the
immunoprecipitated atrial GIRK1-CIR complex (I
; Fig. 2B). The observed binding was due to an
interaction between G
and the channel as determined by
competition with antigenic peptide. As shown in Fig. 2B, the presence of this peptide resulted in a
significant decrease in G
binding. Unlabeled G
similarly decreased the level of binding of labeled G
,
demonstrating specificity of
I-G
binding in the
concentration range under study. Finally, consistent with results from
electrophysiological experiments, the presence of excess G
-GDP,
but not G
-GTP
S, prevented an interaction between G
and I
(Fig. 2B). We conclude that
G
binds directly and specifically to native cardiac
I
. Given the previous reports of I
stimulation by G
-GTP
S(18) , we tested whether
I-G
(GDP- or GTP
S-bound) interacted physically
with immunoprecipitated I
. The amount of
I-G
that bound to immunoprecipitates was
insignificant (
100-fold less) in relation to the amount of bound
G
(data not shown).
The binding constant for the
interaction between G and I
was determined by
competition between unlabeled and labeled G
for channel
binding sites(17) . The binding data were most simply and
adequately fit by a model with a single type of binding site. The
G
binding constant to cardiac I
immunoprecipitated by aCIRN2 was 55 nM (Fig. 2C). Thus, the affinity of the channel for
G
determined in this binding assay was
10-fold lower
than that suggested by results from electrophysiological experiments (Fig. 1B). Since this discrepancy could potentially be
explained by antibody interference with G
binding, we also
determined the G
binding constant to the channel using an
immunoprecipitating antibody targeting the carboxyl terminus of the
GIRK1 subunit of I
instead of the CIR subunit
(aCsh(3) ). The G
binding affinity to aCsh and aCIRN2
immunoprecipitates were virtually identical (Fig. 2C),
indicating that G
binding was not affected by the
immunoprecipitating antibody. Note that Lubrol PX, even at low
concentrations (0.1%), significantly inhibited G
binding,
consistent with the inhibition by Lubrol PX of G
-induced
I
activation observed in patch-clamp
experiments(19) .
Given the results of the binding studies
between G and native cardiac I
, the next
logical step was to determine whether GIRK1, CIR, or both subunits
interacted with G
. Since the subunit-specific antibodies did
not affect the binding of G
to cardiac I
, they
were used to study binding of G
to GIRK1 and CIR subunits
expressed and isolated from Sf9 cells. The control experiments used to
confirm specificity of G
binding to cardiac I
(see Fig. 2B) were also performed for the
individually expressed subunits to assess the significance of any
observed interactions. Both recombinant GIRK1 and CIR subunits
demonstrated specific binding to G
(Fig. 3, A and B). There was no evidence for cooperativity in the
binding of G
to either recombinant subunit; the data were
well fit to a model with a single type of binding site. The apparent
affinity of G
for CIR was almost identical to that for
cardiac I
(K
= 50
nM; Fig. 3C) and slightly lower for GIRK1 (K
= 125 nM).
Despite the widespread electrophysiological evidence for
membrane-delimited G protein activation of ion channels(20) ,
no biochemical evidence has been presented for a direct interaction
between G protein subunits and channel proteins for two major reasons.
First, in comparison to other G protein effectors such as adenylyl
cyclase, cGMP phosphodiesterase, and phospholipase C, channel
proteins are of lower abundance in cells. Ion channels of specific
subtypes number only a few thousand per cell. Second, the most dramatic
example of a G protein-regulated ion channel is I
, a
member of a class of ion channels only recently
cloned(8, 21) . The generation of immunoprecipitating
antibodies to the I
channel subunits, GIRK1 and CIR,
enabled us to develop an assay for G
binding. The present
work shows that G
binds CIR, GIRK1, and the native cardiac
channel with similar affinities.
There are two findings in the
current study that are discrepant with the electrophysiological data.
The concentration of G eliciting half-maximal I
activity was
5 nM in inside-out atrial patches,
while the calculated G
binding constant for the solubilized
channel was
50 nM. This difference could be simply due to
the presence of the higher concentration of CHAPS in the binding
reaction than in the electrophysiological experiments (1.6 versus 0.13 mM). On the other hand, G
is hydrophobic
and when added to an inside-out patch might concentrate in the
membrane, giving a higher G
concentration in the vicinity of
the channel compared with the bath concentration. Thus, such functional
studies might overestimate the real affinity of G
for
I
. Our electrophysiologic data were fit with a Hill
coefficient of 1.5, suggesting mild cooperativity (but see (7) ). However, we did not observe cooperativity in G
binding to the channel or to its individual subunits. It is possible
that detergent solubilization of the channel could influence
interactions between G
binding sites on the channel. Our
current hypothesis is that one G
binds each GIRK1 or CIR
subunit of the I
heteromultimer to activate the channel.
We have shown that I is gated by G
, not
G
(1, 2) , that I
is a
heteromultimer of GIRK1 and CIR inward rectifier K
channel subunits(3) , and that G
, not G
,
directly binds both subunits of cardiac I
. We have not
localized the binding sites for G
on the individual GIRK1 or
CIR subunits, but G
has been shown to associate with a fusion
protein containing the full carboxyl-terminal residues of
GIRK1(16) . Interestingly, our studies indicate that the
antibodies raised against this entire region do not interfere with the
observed binding of G
to cardiac I
. Initially,
the obvious candidate region for G
binding in GIRK1 was the
extreme
150 carboxyl-terminal amino acids, since this domain was
not present in the G protein-insensitive IRK (Kir 2.0) or ROMK (Kir
1.0) families. However, CIR, which binds G
even better than
GIRK1, has no corresponding region. Comparisons of the GIRK (Kir 3.0)
family (including CIR) carboxyl-terminal regions do not reveal any
unique GIRK family similarities, which might suggest a functional
G
-binding domain. In contrast, the amino termini of the GIRK
family contain scattered conserved amino acids not found in ROMK and
IRK subfamilies. Additional structure/function studies will address
these issues.