From the Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, New York, New York 10029
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ABSTRACT |
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Activation of heterotrimeric GTP-binding (G)
proteins by their coupled receptors, causes dissociation of the G
protein and
subunits. G
subunits
interact directly with G protein-gated inwardly rectifying
K+ (GIRK) channels to stimulate their activity. In
addition, free G
subunits, resulting from
agonist-independent dissociation of G protein subunits, can account for
a major component of the basal channel activity.
Using a series of chimeric constructs between GIRK4 and a
G Signaling through GTP-binding (G) proteins depends on dissociation
of the heterotrimer G Five members of the G protein-gated inwardly rectifying K+
(GIRK1-5) channel subfamily have been reported thus far (3-8). The
presumed topology of these channels includes a cytoplasmic N terminus
(~90 amino acids), followed by two transmembrane domains with the
"ion selectivity" P-region in between (~100 amino acids) and
ending with a long cytoplasmic C terminus (over 200 amino acids) (3,
9). GIRK channels can function as highly active heteromultimers
(pairing of GIRK1 with any other subtype) or low to moderately active
homomultimers (GIRK2-5) (for review, see Ref. 10). Mutations at a
specific position within the P-region of these channels ("P-region
mutants", e.g. GIRK4-S143T) greatly enhance the activity
of homomultimers (11, 12). Use of these highly active point mutants
simplifies the experimental design of structure-function studies and
allows assessment of the relative contributions of each of the two
subunits in the heteromultimeric complex (12).
Several studies have demonstrated direct binding of G The We sought to identify those residues of GIRK critical for transducing
effects of the G Human homologs of GIRK1 and GIRK4 (GenBankTM
accession numbers U39195 and U39196) (7) or their point mutated active counterparts (GIRK1-F137S or GIRK1* and GIRK4-S143T or GIRK4*), subcloned in the pGEMHE plasmid vector (23), were used as described previously (11, 12). The chimeric cDNA constructs were produced by
splicing by overlapping extension polymerase chain reaction (24).
Polymerase chain reactions, using Vent DNA polymerase, were performed
for only 15 cycles to avoid errors. Point mutations were generated
using the Quickchange site-directed mutagenesis kit (Stratagene). The
sequence of all constructs was confirmed by automated DNA sequencing
(Sequencing facility, Cornell University, Ithaca, NY). The All constructs were linearized with Nhel and cRNAs were
transcribed in vitro using the "message machine" kit
(Ambion). RNAs were electrophoresed on formaldehyde gels and
concentrations were estimated from two dilutions using an RNA marker
(Life Technologies, Inc.) as a standard.
Xenopus oocytes were surgically extracted, dissociated,
defolliculated by collagenase treatment, and microinjected with 50 nl
of a water solution containing the desired cRNA. Unless otherwise indicated, we used the following approximate quantities: GIRK channel
subunits, 1.0 ng/species; IRK1 channel, 0.25 ng; m2 receptor, 1.0 ng;
Single-channel activity was recorded on devitellinized oocytes under
the cell-attached mode of standard patch-clamp methods (26), as
described previously (27). The pipette solution contained 96 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4. The bath solution was composed of 96 mM KCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.4. 100 µM gadolinium was also
included in the pipette solution. G Recombinant bovine G GIRK4* Unlike IRK1 Is G
Expression of GIRK4* in oocytes led to large basal and ACh-induced
currents (Fig. 1, A and
C). Coexpression of either A Minimal Chimera between GIRK4* and IRK1 with a Defect in
Agonist-induced Responses--
We constructed chimeras between GIRK4*
and IRK1 (Fig. 2, left). We
screened for minimal segments of GIRK4* that when replaced by the
corresponding IRK1 regions impaired sensitivity to
G
Chimeras were named for the IRK1 segment replacing the corresponding
GIRK4 region. We first replaced the full C terminus of GIRK4* with that
of IRK1 (GIRK4*(IRKV179-I428)). This chimera showed intact
basal but impaired agonist-induced currents, consistent with a previous
report (22). Huang et al. (15) found the
GIRK1(Glu318-Pro462) segment to be a minimal
G An Agonist-insensitive Chimera with Intact
G A Point Mutation Sufficient to Specifically Impair Agonist-induced
Currents without Affecting the G
We next tested the G
Oocytes coexpressing GIRK4*(L339E), G The C Terminus of GIRK4(L339E) Shows Decreased Binding to the
G
As shown in Fig. 5 (middle and bottom panels),
the C termini of GIRK4 and GIRK4(L339E) were able to bind
G Wild-type GIRK1/GIRK4 Subunits Contribute Equally to the
Agonist-induced Activity of Heteromultimeric Channels--
To
determine whether the effect seen with the L339E mutant was specific to
the GIRK4* subunit, we mutated the corresponding amino acid residue in
GIRK1, L333E. We tested for G
Similar results were obtained with the GIRK1*(L333E) mutant as with the
GIRK4*(L339E). Again, although ACh-induced currents were impaired by
the mutation, the basal currents were reduced by Gi
Fig. 6B shows that GTP
We next sought to determine the relative contribution of wild-type GIRK
subunits to agonist-induced activation in heteromultimeric channels
(Fig. 6C). We introduced the Leu to Glu mutations at the 333 and 339 positions of the wild-type GIRK1 and GIRK4 subunits, respectively. We compared basal and agonist-induced currents of GIRK1/GIRK4 heteromultimeric channels, composed of both wild-type, both
Leu to Glu mutants, and each wild-type to mutant combination. Our
results suggest that each of the wild-type subunits contribute equally
to agonist-induced activity, because heteromultimeric channels
containing either Leu to Glu mutants displayed reduced agonist-induced
sensitivity. Moreover, heteromultimeric channels, where both the
subunits contained the Leu to Glu substitution showed significantly
impaired agonist-induced currents. Thus, these results confirmed the
importance of the residue for receptor-stimulated currents in
heteromultimeric channels.
Mutation of the Critical Leu Residue Does Not Distinguish among
Channel Interactions with Specific G
G Since Soejima and Noma (29) first reported the membrane-delimited
nature of the atrial muscarinic K+ channel, the mechanism
of G protein gating of ion channels has received great attention. Over
a decade ago, G protein-gated inwardly rectifying K channels provided
the first example of a G Biochemical studies from several groups have pointed to interaction of
G In this study, we screened the C terminus of GIRK4* for residues that
control channel activity. We made chimeras that replaced specific
sections of GIRK4* with those from the G protein-insensitive channel,
IRK1. Expression of a minimal chimera, the
GIRK4*(IRKL316-Y341), resulted in normal basal currents
that did not respond to ACh when coexpressed with hm2 receptors.
Expression of exogenous G Of all the amino acid differences between IRK1 and GIRK4* contained in
this chimera, only the mutant GIRK4*(L339E) retained all properties of
the chimera. This mutant displayed G Basal currents from the GIRK4*(L339E) channel were inhibited by
coexpression of G How does GIRK4*(L339E) impair specifically agonist-induced stimulation?
In the simplest model, free G GIRK1*(L333E) channels displayed similar properties to GIRK4*(L339E).
Again, basal currents from this mutant channel were sensitive to
G Biochemical evidence has suggested multiple binding sites in the C- and
N-terminal segments of GIRK channels (15, 19, 30). The multiplicity of
G-insensitive K+ channel, IRK1, we have
identified a critical site of interaction of GIRK with
G
. Mutation of Leu339 to Glu within this
site impaired agonist-induced sensitivity and decreased binding to
G
, without removing the G
contribution to basal currents. Mutation of the corresponding residue
in GIRK1 (Leu333) resulted in a similar phenotype. Both the
GIRK1 and GIRK4 subunits contributed equally to the agonist-induced
sensitivity of the heteromultimeric channel. Thus, we have identified a
channel site that interacts specifically with G
subunits released through receptor stimulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
into the
G
-GTP and G
subunits. Direct
interactions of G
or G
(or both) with
effector proteins transduces the external signal into an intracellular
response. Atrial potassium (K+) channels, the first example
of a G
effector (1), are responsible for the
acetylcholine(ACh)1-induced
reduction in heart rate during vagal activity (2).
subunits to entire GIRK proteins (13) or to segments of channel
subunits (14-19). Although G
subunits can interact directly with both N and C termini, interactions with the C terminus of
the channel were shown to be the strongest (14, 15). In addition, the N
terminus also binds to G
subunits alone (14) or to the
G
heterotrimer (14, 18).
subunits of G proteins activate not only native GIRK
heteromultimers (1, 6), but also recombinant hetero- or homomultimeric
GIRK channels (7, 20). There is no qualitative difference in the
G
sensitivity of P-region homomultimeric mutants
versus heteromultimeric channels (12). In contrast, the
inwardly rectifying K+ channel IRK1 (21) is
G
insensitive (22), despite its high degree of
similarity to the five members of the GIRK subfamily.
subunits. Our strategy was to
generate chimeras between the GIRK4(S143T) (referred to as GIRK4*) and
IRK1 channels, and screen for differences in
G
-dependent function and binding to
G
. Mutagenesis at a single site, namely GIRK4(L339E),
reduced binding to G
and impaired agonist-induced
activity, but left intact the G
dependence of the
basal activity. Thus, we have identified a site on an effector protein
that interacts specifically with G
released through
receptor stimulation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ARK-PH
construct (amino acids 452-689) was altered to incorporate the 15 N-terminal residues of Src for membrane targeting. This construct,
generously provided by Dr. Eitan Reuveny, was altered and subcloned
into pGEMHE.
2-adrenergic receptor, 2.0 ng; G
and G
subunits, 1.0 ng; G
subunit, 1.0 ng;
ARK-PH, 1.0 ng.
G
2 and G
2 were used in all
G
coexpression experiments, unless otherwise
indicated. Oocytes were incubated for 3 days at 19 °C. Whole oocyte
currents were then measured by conventional two-microelectrode voltage
clamp with a GeneClamp 500 amplifier (Axon Instruments). Agarose
cushion microelectrodes were used with resistances between 0.1 and 1.0 megaohms (25). Oocytes were constantly superfused with a high potassium
solution having (in mM): 91 KCl, 1 NaCl, 1MgCl2, 5 KOH/HEPES (pH 7.4). To block or activate
currents, the oocyte chamber was perfused with solutions of the same
composition with 3 mM BaCl2 or 5 µM ACh. Typically oocytes were held at 0 mV
(EK), and currents were constantly monitored by
500 ms pulses to a command potential of
80 mV for 200 ms followed by
a step to +80 mV for another 200 ms, and the cycle was repeated every 2 s. Periodically, a protocol was applied with a command potential from
100 to +100 mV with 10 mV increments. Current amplitudes were
measured at the end of the 200 ms pulse at each potential. In such
manner, control currents were evaluated 2-5 min after impaling the
oocytes just before application of ACh, ACh-activated currents were
evaluated at the peak of the response to ACh, and Ba2+-insensitive currents were evaluated once steady-state
inhibition was achieved, 1-3 min after application of 3 mM
Ba2+. Basal current is the difference between control and
Ba2+-insensitive currents, and ACh-induced (or
ACh-sensitive) current the difference between ACh-activated and control
currents. Error bars in the figures represent mean ± S.E. Each
experiment shown or described was performed on 3-5 oocytes of the same
batch. A minimum of 2-3 batches of oocytes were tested for each
experiment shown.
purified from
bovine brain was used in the inside-out experiments (gift from Dr. John
D. Hildebrandt). Recordings were performed at a holding membrane
potential of
80 mV. Recordings were performed using the EPC-9
patch-clamp amplifier and the PULSE/PULSEFIT (v. 7.6) data acquisition
software (Heka Electronik, Lambrecht, Germany). Data were stored on the
hard disk of a PC compatible computer, and single channel analysis made
use of TAC (v 2.6.1) software (Skalar Instruments, Inc., Seattle, WA).
The sampling rate was 4 kHz for most recordings. Activity expressed as
NPo (N, number of channels in the patch; Po, probability of opening)
was calculated by integrating the current traces over 30-60 s
intervals and dividing by the unitary current.
1
2 subunits were purified from
Sf9 cells infected with baculoviruses encoding for
1,
2, and His6-
i1 as described by
Kozasa and Gilman (32). cDNAs encoding the C termini of GIRK4,
GIRK4(L339E), or IRK1, and the PH domain of
ARK were generated by
polymerase chain reaction and cloned in frame with the GST coding
sequence in pGEX-4T-3 (Amersham Pharmacia Biotech). The resulting
polymerase chain reaction fragments coded for: amino acids 184-419 for
GIRK4, GIRK4(L339E), amino acids 177-428 for IRK1; and amino acids
546-670 for
ARK. Expression of fusion proteins was induced by 0.1 mM isopropyl-1-thio-b-D-galactopyranoside at
37 °C for 2 h, and the fusion proteins were purified using glutathione 4B-Sepharose beads. The binding assay of
G
to the fusion proteins was performed as described
by Huang et al. (14). Briefly, 1.0 µM GST
fusion protein and 0.1 µM G
were
incubated in phosphate-buffered saline, 0.1% lubrol and
glutathione-Sepharose beads at 4 °C for 30 min. After washing three
times with 200 volume of phosphate-buffered saline with 0.01% lubrol,
the bound proteins were eluted from beads by heating in protein sample
buffer at 70 °C for 10 min and were then electrophoresed in a 12%
SDS-polyacrylamide gel electrophoresis. GST fusion proteins were
visualized by Coomassie staining, and G
1 was detected by
immunoblotting using a G
antibody (Santa Cruz
Biotechnology) and visualized with ECL (Amersham). Densitometry was
used to quantify the relative amounts of bound G
.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sensitive--
We
compared G
sensitivities of basal and agonist-induced
currents between the highly active GIRK4* and IRK1 channels. All
experiments were carried out by expression in Xenopus
oocytes. In whole-cell experiments, G
sensitivity, in
the presence of coexpressed G protein-coupled receptor, was assessed by
(a) K+ current responses to agonist stimulation,
(b) coexpression of channels with proteins such as Gi
subunits or the PH domain of
ARK that can act as "sinks" for
endogenous G
subunits, and (c)
coexpression of channels with exogenous G
subunits.
ARK-PH or Gi
1 led to a
significant reduction in basal currents. However, oocytes coexpressing
Gi
1, rather than
ARK-PH, displayed ACh-induced currents. This
result is consistent with the interpretation that Gi
1, and not
ARK-PH, bound to endogenous G
may be available for
receptor-mediated activation. Coexpression of GIRK4 with exogenous
G
-enhanced agonist-independent K+
currents while preserving the ACh-induced response (as in Ref. 12).
These results indicate that both the GIRK4* basal and agonist-induced currents are largely mediated by the G
subunits. In contrast, oocytes injected with IRK1 exhibited no ACh-induced currents
and did not respond to coexpression with Gi
1,
ARK-PH, or
G
(Fig. 1, B and C).
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Fig. 1.
G
sensitivity of the GIRK4* and IRK1 inwardly rectifying
channels. A, current-voltage (I-V) relationships of basal and
agonist-induced currents of GIRK4(S143T), designated as GIRK4*,
coexpressed with human muscarinic receptor 2 (hm2). Coexpression with
ARK-PH or Gi
1 or the stimulatory G
subunits is
indicated. Squares represent basal currents, whereas
circles indicate total currents in the presence of ACh.
Points in the I-V curves represent mean ± S.E. for 3-5 oocytes
within this batch of oocytes. B, current-voltage
relationships of IRK1 under identical experimental conditions, as shown
in A. C, basal and agonist-induced currents of
the experiments described in A and B at
80 mV.
Bars represent mean ± S.E. for three to five separate
experiments.
.
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Fig. 2.
Chimeras between GIRK4* and IRK1 channels
reveal a region important in agonist-induced stimulation of
K+ currents. Left, schematic of chimeric
constructs between GIRK4* and IRK1. Specific segments of IRK1
(black regions) were used to replace corresponding segments
of GIRK4* (white regions). Each chimera was named according
to the IRK1 segment (identified by the first and last IRK1 amino acid
of the segment), which replaced the corresponding segment of GIRK4*.
Note that the corresponding numbers, defining the limits of the grafted
segments, are shifted (6 to 7 numbers) as determined by the alignment
of the two sequences. Right, basal and agonist-induced
currents at 80 mV of GIRK4*, IRK1, and the five chimeras.
Asterisks note significant differences in ACh-induced
currents of the particular chimeras from the GIRK4* control (GIRK4*,
10.5 ± 2.98 µA, n = 4;
GIRK4*(IRKL316-Y341),
0.62 ± 0.52 µA,
n = 4; p < 0.05).
binding region. From an alignment of the GIRK1 and
GIRK4 primary amino acid sequences, residue GIRK1(Glu318)
corresponds to GIRK4(Asp324). Thus, we tested the response
of the chimera GIRK4*(IRKL316-I428) that replaced the
GIRK4(Met323-Val419) region with the
corresponding IRK1 segment. Again this chimera exhibited intact basal
but impaired agonist-induced currents. To narrow the region responsible
for the aberration of the GIRK4* agonist-induced currents, we
constructed and tested three additional chimeras
GIRK4*(IRKL316-Y341), GIRK4*(IRKS342-K365), and
GIRK4*(IRKY366-I428). All three chimeras displayed
intact basal currents. However, the response of
GIRK4*(IRKL316-Y341) was impaired to agonist. These results
suggest that differences between the two channels in this region,
GIRK4(Met323-Tyr348) and
IRK1(Leu316-Tyr341), may be important in their
differential sensitivity to G
. This is unlike the
downstream regions where differences were without effects on
G
sensitivity.
-mediated Basal Currents--
The current resulting
from expression of the chimera GIRK4*(IRKL316-Y341) had
intact basal currents but impaired agonist-induced responses. To test
its sensitivity to G
, the
GIRK4*(IRKL316-Y341) chimera was coexpressed with Gi
1 or
ARK-PH. A significant reduction of basal currents was obtained,
similar to the GIRK4* control (Fig.
3A). Yet, this chimera
differed from GIRK4* (see Fig. 1) in that its expression alone or with
Gi
or G
resulted in impaired ACh-induced currents.
This result further supports the conclusion that this chimeric channel
is defective in producing agonist-induced currents. Coexpression with
G
did not stimulate basal levels of activity. Because
the G
dependence of the basal currents was intact in
the GIRK4*(IRKL316-Y341) chimera, it is likely that other
regions may be involved in G
mediation of basal
currents.
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Fig. 3.
G
sensitivity of basal currents of the agonist-insensitive chimera
and point mutants. A, effects on the basal currents (at
80 mV) of the agonist-insensitive chimera
GIRK4*(IRKL316-Y341) coexpressed with G
,
ARK-PH, or Gi
1. ACh responses were impaired in all groups
p < 0.005, n = 3-5. The basal
currents of GIRK4*(IRKL316-Y341) coexpressed with
ARK-PH
or Gi
1 were also significantly reduced p < 0.005, n = 3-5. B, currents (at
80 mV) from
mutants resulting from substitution of amino acids within
GIRK4*(M323-Y348) (corresponding to
GIRK4*(IRKL316-Y341)), which differ between GIRK4 and IRK1.
Each of eleven mutations were introduced into the context of the GIRK4*
backbone. Asterisks denote significant reduction in
ACh-induced currents (p < 0.005, n = 3).
Contribution to
Basal Activity--
We proceeded to test which of the distinct
residues within the identified region of the GIRK4* and IRK1 channels
were responsible for their differences in sensitivity to
G
. Eleven point mutations were made in which residues
in the Met323-Tyr348 region of GIRK4* were
mutated to the corresponding residues found in the
Leu316-Tyr341 region of IRK1 (Fig.
3B). Mutant names refer to the position and amino acid of
GIRK4 that was mutated to the corresponding IRK1 residue. Only
GIRK4*(L339E) showed impaired agonist-induced responsiveness, mimicking
the responses obtained with the GIRK4*(IRKL316-Y341) chimera.
sensitivity of the basal
currents of GIRK4*(L339E), and compared them with that of the GIRK4* control in the same batch of oocytes.
, Gi
1, or
ARK-PH behaved similar to the chimera
GIRK4*(IRKL316-Y341), demonstrating an intact
G
-mediated basal current component. (Fig. 4A). Inside-out patch
recordings from oocytes expressing the mutant and control channels were
performed to test their responses to G
subunits. Fig.
4B (left) compares activity from one batch of
oocytes expressing GIRK4* and GIRK4*(L339E) channels. Perfusion of
inside-out patches with purified G
was ineffective in
stimulating GIRK4*(L339E) activity compared with control GIRK4*.
Stimulation of currents by endogenous G proteins through GTP
S
application gave similar results as the application of purified
G
(data not shown, n > 3).
Regardless of their sensitivity to G
, control or
mutant channels responded to a similar degree to intracellular
Na+ ions (27), thus providing a positive control for gating
by Na+ ions. These inside-out patch responses were
consistent with the whole-cell data for GIRK4*(L339E). Perhaps the lack
of stimulation of whole-cell currents by G
coexpressed with GIRK4*(L339E) reflects maximal basal currents for this
mutant. To examine this possibility, we coexpressed
ARK-PH in
oocytes (same batch as the experiments in Fig. 4B,
left) with GIRK4* or GIRK4*(L339E) channels. We perfused
inside-out patches from such oocytes with G
purified
from bovine brain (Fig. 4B, right). Inside-out patches of oocytes coexpressing GIRK4*(L339E) and
ARK-PH
convincingly responded to perfusion with exogenous G
,
presumably recovering the
ARK-PH inhibition of the basal currents
seen in the whole-cell experiments. However, these responses were
significantly smaller than those of the control GIRK4*. In all cases,
GTP
S application failed to stimulate channel activity by activating endogenous G proteins, serving as a positive control for
ARK-PH effectiveness (data not shown, n > 3). Through these
experiments we conclude that the GIRK4(L339E) mutation selectively
impairs agonist-induced G
-mediated responses.
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Fig. 4.
Comparison of
G sensitivity of
GIRK4* and GIRK4*(L339E) in whole-cell and inside-out patch
experiments. A, two-electrode voltage clamp experiments
plotting currents (at
80 mV) of GIRK4* and GIRK4*(L339E) channels
coexpressed with G
,
ARK-PH, or Gi
1. ACh
responses were impaired in the GIRK4*(L339E) groups, p < 0.005, n = 3-6. The basal currents of GIRK4*(L339E)
coexpressed with
ARK-PH or Gi
1 were also significantly reduced,
p < 0.005, n = 3-6. B,
(left) inside-out patches from oocytes expressing the
control or point mutant GIRK4* channels. Responses to patch perfusion
with G
or Na+ are shown for a
representative patch and a number of patches tested within this batch
of oocytes. G
increased channel activity
significantly compared with control for GIRK4* (p < 0.005, n = 3) but not for GIRK4*(L339E)
(n = 4); (right) inside-out patches from
oocytes coexpressing the control or point mutant GIRK4* channels and
ARK-PH. Responses to patch perfusion with G
or
Na+ are shown for a representative patch and a number of
patches tested within this batch of oocytes. G
increased channel activity significantly compared with control for
GIRK4* and GIRK4*(L339E) (p < 0.005, n = 4). The increase in channel activity in response to
G
was significantly less in GIRK4*(L339E) compared
with GIRK4* (p < 0.005, n = 3-4).
Subunit--
To determine the effects of the
GIRK4(L339E) mutation of the C terminus on G
binding, we constructed and purified GST fusion proteins
containing the C termini of GIRK4 (GIRK4C), GIRK4(L339E),
(GIRK4- (L339E)C) and IRK1 (IRK1C), or
ARK-PH. GST fusion
proteins were expressed in bacteria and purified (Fig. 5, top). In vitro
binding assays were performed with the recombinant bovine
G
1
2 subunits purified from Sf9 cells.
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Fig. 5.
The Leu339 mutation of the GIRK4
C terminus causes a significant reduction in
G binding. GST
fusion proteins were purified using glutathione 4B-Sepharose beads and
were detected by Coomassie staining (top). Purified GST
fusion protein were incubated with G
and
glutathione-Sepharose beads. Following wash, the bound proteins were
released from the beads by heating in protein sample buffer and were
separated by SDS-polyacrylamide gel electrophoresis. G
1
was detected by immunoblotting with anti-G
antibody. The
position of the
subunit of G
is indicated by the
arrow. GST was used as negative control, and GST-
ARK-PH
as positive control (middle). Density scanning was used to
quantify the relative amounts of bound G
(bottom). The results shown represents mean ± S.E. for
four separate experiments. *, p < 0.01 compared with
the GST-GIRK4C group.
as compared with negative controls (GST and IRK1C)
and a positive control (
ARK-PH). GIRK4(L339E)C binding to
G
was significantly reduced. These results suggest
that the critical Leu of GIRK channels, and perhaps neighboring
residues, directly interacted with G
subunits.
Additionally, because GIRK4(L339E)C has reduced but measurable
binding to G
, it is likely that additional C-terminal
G
binding sites exist, which contribute to the
G
dependence of basal currents.
sensitivity of this
point mutant in the context of the highly active homomultimer
GIRK1(F137S) (see Refs. 11 and 12; referred to as GIRK1*).
1 and
ARK-PH
(Fig. 6A,
right).
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Fig. 6.
Mutation of the critical Leu residue in
GIRK1* or wild-type heteromultimers GIRK1/GIRK4 also impairs
agonist-induced responses. A, two-electrode voltage
clamp experiments plotting currents (at 80 mV) of GIRK1* and
GIRK1*(L333E) coexpressed with G
,
ARK-PH, or
Gi
1. B, representative records of inside-out patches show
the response of GIRK1* and GIRK1*(L333E) currents to GTP
S
application. C, Leu to Glu substitutions of the wild-type
GIRK1 or GIRK4 (not in the context of GIRK1* or GIRK4*) were performed.
Coexpression of each mutant with the complementary wild-type subunit
and of both mutant subunits. The responses to ACh were reduced
significantly for all mutant bearing heteromultimers
GIRK1(L333E)/GIRK4, GIRK1/GIRK4(L339E), and
GIRK1(L333E)/GIRK4(L339E) compared with wild-type
GIRK1/GIRK4, p < 0.005, n = 4.
S application to inside-out patches
expressing GIRK1* or GIRK1*(L333E) resulted in ~42-fold increase in
GIRK1* activity but caused no increase in the current of GIRK1*(L333E) (n = 5).
Subunits or
Signaling through Specific Receptors--
Yeast two-hybrid experiments
have shown that G
1 and G
2 interact with
the N terminus of GIRK1 more strongly than do G
3-5
(19). To determine whether C-terminal mutation of the critical Leu
residue could have altered the ability of the channel to interact with
specific G
subunits, we coexpressed G
1,
G
2, or G
3 with G
2 and
GIRK1/GIRK4 or GIRK1(L333E)/GIRK4(L339E) heteromultimers. All
G
combinations stimulated wild-type basal currents
(2-4-fold, n = 3). When tested with the mutated channel subunits, G
1-3
2 subunits failed to stimulate basal currents (n = 3). These results suggest that
mutation of the critical Leu residue does not exert its effects by
altering the specificity of channel/G
1-3 interactions.
However, possible changes in the specificity of Leu mutant channels
with the G
4 or G
5 subunits that were not
tested cannot be ruled out.
subunits released from Gs
subunits
by
2-adrenergic receptor stimulation activate GIRK channels
expressed in Xenopus oocytes (28). To test whether the
critical Leu residue is involved in G
signaling by
receptors other than m2, we coexpressed
2-adrenergic receptor and
Gs
subunits with GIRK1/GIRK4 or GIRK1(L333E)/GIRK4(L339E)
heteromultimers. Isoproterenol-induced currents were obtained after
expression of wild-type heteromultimers (
7.65 ± 2.17 µA at
80 mV, n = 3) but not with mutants (
0.17 ± 0.09 µA at
80 mV, n = 3). These results suggest
that the G
released after activation of these two
different receptors interact in a similar fashion with the critical
GIRK Leu residue.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
controlled signaling
pathway. Yet, despite intense efforts, many questions remain unanswered
regarding specific sites of interaction between the channel and
G
.
with the C and N termini of these channels. Specifically, Huang et al. (14), using deletion mutagenesis, found that deletion of the GIRK1(Val273-Pro354)
segment reduced G
binding of the remaining C-terminal fragment. In subsequent studies, Huang et al. (15)
determined the GIRK1(Glu318-Pro462) segment as
a minimal G
binding region. Kunkel and Peralta (16),
using a combination of chimeras and deletion mutations, reported the
GIRK1(Thr290-Tyr356) region to be important in
interactions with G
.
did not enhance basal
currents in this chimera. Yet, basal currents were inhibited by
coexpression of Gi
1 and
ARK-PH.
-sensitive basal
currents that were not ACh-sensitive and did not respond to exogenous
G
. Inside-out patch currents from oocytes expressing
GIRK4*(L339E) and hm2 receptors were significantly smaller in response
to G
or GTP
S. Binding of the
G
subunits to the GIRK4 C terminus bearing the L339E
mutation was significantly reduced. Within the broader boundaries
suggested by others (14-16), the region surrounding
Leu339 is a G
binding site with critical
functional consequences.
sinks, such as the
ARK-PH.
Because binding of the L339E mutant of the GIRK4 C terminus was not
abolished and because basal currents of GIRK4*(L339E) could be
inhibited by
ARK-PH or Gi
1, it is likely that additional
G
binding sites contribute to basal channel activity.
Moreover, because G
perfusion of inside-out patches
activated GIRK4*(L339E) channels only when they were coexpressed with
ARK-PH, it is likely that this activation reflected reversal of the
ARK-PH inhibited basal currents. We hypothesize that the basal
binding sites may be high affinity and saturated in both the whole-cell
and inside-out patch experiments. Recent evidence has suggested another
region of GIRK4 (Ser209-Arg225) capable of high
affinity binding to G
(30). It is possible that such
a site accounts for part or all of the basal channel activity.
would be bound to high
affinity basal sites. The GIRK4*(IRKL316-Y341) chimera and
the GIRK4*(L339E) mutant may impair a low affinity binding of this
region to G
subunits. Normally, agonist-induced liberation of G
subunits would increase the local
free G
concentration, allowing interaction with a low
affinity site, encompassing GIRK4*(Leu339), and leading to
stimulation of channel activity. Further work will be required to test
this hypothesis.
, but no ACh-induced currents could be detected. Double mutations in heteromultimeric
GIRK1(L333E)/GIRK4(L339E) channels expressed in oocytes
showed similar properties to the highly active homomultimeric mutants
discussed above. Furthermore, mutation of both channels in a
heteromultimer was required for the ACh-insensitive phenotype, whereas
reduced agonist-induced currents were obtained with one or the
other of the two subunits mutated. These results suggest that there is
an equivalent contribution of GIRK1 and GIRK4 to
G
-mediated ACh-induced activity. Additionally,
coexpression of different G
combinations or different
receptors such as the
2-adrenergic receptor did not alter the unique
properties of these mutant channels. This suggests that signaling
through different receptors and by different G
combinations activates the channel through conserved interactions.
binding sites with effector proteins is in
agreement with the finding that the G
-phosducin co-crystals show multiple sites of interaction between the two proteins
(31). Our data combine biochemical with functional evidence for more
than one G
binding site on the channel. Surprisingly,
distinct functional roles could be assigned to multiple binding sites;
one designed to interact with G
released from
receptor stimulation, whereas additional site(s) may interact with free
G
to yield basal activity. Thus, these results suggest that for the K+ channel the multiplicity of
interactions may subserve distinct functional roles.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Eitan Reuveny for
generously sharing with us the modified version of the ARK-PH
construct and to Dr. Maureen Linder for graciously guiding us with the
G
purification. We thank Drs. David Clapham and
Robert Margolskee for critical review of the manuscript, Grigory
Krapivinsky for advice with the binding studies, and Mariana Max, Eitan
Reuveny, and Ming Ming Zhou for helpful discussions.
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FOOTNOTES |
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* This work was supported by Grants from the National Institutes of Health (HL54185) and American Heart Association (National Center 96011620) (to D. E. L.).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.
These authors contributed equally to this work.
§ Associate of the Howard Hughes Medical Institute.
¶ Supported by National Institutes of Health Training Grant (HL07824).
To whom correspondence should be addressed: Dept. of
Physiology and Biophysics, Box 1218, Mount Sinai School of Medicine, CUNY, 1 Gustave L. Levy Pl., New York, NY 10029-6574. Tel.:
212-241-6285; Fax: 212-860-3369; E-mail:
logothetis{at}msvax.mssm.edu.
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ABBREVIATIONS |
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The abbreviations used are:
ACh, acetylcholine;
GIRK, G protein-gated inwardly rectifying K+;
PH, pleckstrin homology;
GST, glutathione S-transferase;
GTPS, guanosine 5'-O-(thiotriphosphate).
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REFERENCES |
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