THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 275, No. 46, Issue of November 17, pp. 36211–36216, 2000
Printed in U.S.A.
Functional and Biochemical Evidence for G-protein-gated Inwardly
Rectifying Kⴙ (GIRK) Channels Composed of GIRK2 and GIRK3*
Received for publication, August 4, 2000, and in revised form, August 22, 2000
Published, JBC Papers in Press, August 23, 2000, DOI 10.1074/jbc.M007087200
Tanya M. Jelacic‡, Matthew E. Kennedy, Kevin Wickman, and David E. Clapham§
From the ‡Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Foundation, Rochester,
Minnesota 55905 and the Howard Hughes Medical Institute, Children’s Hospital and Harvard Medical School,
Boston, Massachusetts 02115
G-protein-gated inwardly rectifying Kⴙ (GIRK) channels are widely expressed in the brain and are activated
by at least eight different neurotransmitters. As Kⴙ
channels, they drive the transmembrane potential toward EK when open and thus dampen neuronal excitability. There are four mammalian GIRK subunits
(GIRK1– 4 or Kir 3.1– 4), with GIRK1 being the most
unique of the four by possessing a long carboxyl-terminal tail. Early studies suggested that GIRK1 was an integral component of native GIRK channels. However,
more recent data indicate that native channels can be
either homo- or heterotetrameric complexes composed
of several GIRK subunit combinations. The functional
implications of subunit composition are poorly understood at present. The purpose of this study was to examine the functional and biochemical properties of GIRK
channels formed by the co-assembly of GIRK2 and
GIRK3, the most abundant GIRK subunits found in the
mammalian brain. To examine the properties of a channel composed of these two subunits, we co-transfected
GIRK2 and GIRK3 in CHO-K1 cells and assayed the cells
for channel activity by patch clamp. The most significant difference between the putative GIRK2/GIRK3 heteromultimeric channel and GIRK1/GIRKx channels at
the single channel level was an ⬃5-fold lower sensitivity
to activation by G␤␥. Complexes containing only GIRK2
and GIRK3 could be immunoprecipitated from transfected cells and could be purified from native brain tissue. These data indicate that functional GIRK channels
composed of GIRK2 and GIRK3 subunits exist in brain.
The G protein-gated class (Kir 3, GIRK)1 of potassium-selective ion channels are assembled from a small family of subunits. In the nervous system, heart, pancreas, and other tissues, transmitters activate the GIRK channels via an
apparently identical G␤␥-linked signal transduction mechanism. The G protein-linked receptor subtypes that activate
these channels include muscarinic (M2), ␥-aminobutyric acid
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
§ To whom correspondence should be addressed: Howard Hughes
Medical Inst., Children’s Hospital and Harvard Medical School, Enders
1309, 320 Longwood Ave., Boston, MA 02115. Tel.: 617-355-6163; Fax:
617-731-0787; E-mail: [email protected].
1
The abbreviations used are: GIRK, G-protein-gated inwardly rectifying K⫹; PBS, phosphate-buffered saline; GTP␥S, guanosine 5⬘-3-O(thio)triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; NPo,
N is the number of channels in the patch, and Po is the open probability
of the channel.
This paper is available on line at http://www.jbc.org
(GABAB), serotonin (5HT1A), adenosine (P1), somatostatin,
enkephalin (␮, ␬, ␦), ␣2⫺adrenergic, and dopamine (D2) receptors. GIRK channels composed of GIRK1 subunits in combination with either GIRK2, GIRK3, or GIRK4 subunits have indistinguishable single channel conductances, open times, and
G␤␥ sensitivities (1). In contrast, GIRK2, -3, or -4 subunits
expressed alone produce spiky, irregular amplitude channels
with very brief open times (1– 6). GIRK1 subunits alone do not
make functional channels but require co-expression of GIRK2,
-3, or -4 subunits to achieve membrane localization and yield
functional channels that have identical properties to native
cardiac IKACh (7–9). Studies on the GIRK1/GIRK4 channel,
IKACh, have indicated that GIRK4 (and presumably GIRK2 and
GIRK3) contributes determinants that direct the channels to
the membrane (8) as well as cytoplasmic domains that bind
G␤␥ and are important for channel activation (10 –12).
While most brain regions express GIRK1, GIRK2, and
GIRK3 mRNAs (13–15), there are regions where only GIRK2
and GIRK3 mRNA transcripts are present or expression of
GIRK1 is very low. We combined electrophysiological and biochemical approaches to determine if a GIRK heteromultimer
composed of GIRK2 and GIRK3 subunits alone was functional
and if such a channel exists in vivo. GIRK2 and GIRK3 subunits had been previously coexpressed in Xenopus oocyte expression studies (16 –19), but the presumed GIRK2/GIRK3 currents observed were small (⬍0.1 ␮A). In this study, we examine
this subunit combination in mammalian cells. We demonstrate
that GIRK2/GIRK3 forms a functional channel in heterologous
cells and that a GIRK2/GIRK3 complex can be purified from
both heterologous expression systems and native brain tissue.
EXPERIMENTAL PROCEDURES
Cell Culture and Transfection—CHO-K1 cells were grown and maintained in 1:1 Dulbecco’s modified Eagle’s medium/Hamm’s F-12 (Life
Technologies, Inc.) with 10% fetal bovine serum at 37 °C under 5% CO2.
CHO-K1 cells were transfected using TransIt娂-LT1 (Pan Vera Corp.,
Madison, WI) in OptiMEM I medium (Life Technologies, Inc.). The
pGreen Lantern plasmid (Life Technologies, Inc.) was cotransfected
with GIRK-containing pcDNA3 or pcDNA3.1(⫺) expression vectors (Invitrogen, Carlsbad, CA) in a 1:4 ratio as a transfection marker for patch
clamp experiments. GIRK plasmids were co-transfected in a 1:1 ratio.
The GIRK2- and GIRK3-containing plasmids used have been described
previously (1, 20). Transfected CHO-K1 cells were detached from their
dishes with 5 mM EDTA in PBS, resuspended in growth medium, and
transferred to glass coverslips for patch clamp experiments. COS-7 cells
were grown and maintained in Dulbecco’s modified Eagle’s medium
with 10% fetal calf serum at 37 °C in 5% CO2. COS-7 cells were plated
at 2 ⫻ 106 cells/100-mm2 dish the day before transfection using either
LipofectAMINE (Life Technologies) or TransIt娂-LT1.
Electrophysiology—Single channel recordings were made from
patches in the inside-out configuration in symmetrical high K⫹ (140 mM
KCl, 2 mM MgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.2). Patch
electrodes were prepared from borosilicate capillary glass (Warner Instrument Corp., Hamden, CT) and polished to a resistance of 5–12
36211
36212
GIRK2/GIRK3 Channels
megaohms. Patches were activated by application of either 40 ␮M
GTP␥S (Roche Molecular Biochemicals) or 2–90 nM purified bovine
brain G␤␥ (2) to the cytoplasmic face of the patch. Currents were
recorded using an Axopatch 200A amplifier (Axon Instruments, Inc.,
Foster City, CA), filtered at 2 kHz by a four-pole low pass Bessel filter,
and sampled at 10 kHz.
Records of channels in inside-out patches activated by 40 ␮M GTP␥S
were collected at voltages ranging from ⫺100 to ⫺40 mV. Unitary
current values from all patches were averaged, and the slope conductance was determined as the best fit to the mean data. Channel open
times were determined for 10-s records of inside-out patches activated
by 40 ␮M GTP␥S collected at ⫺100 mV. Log analysis (21) was used for
determination of channel mean open time (␶o).
The concentration dependence of channel activation by G␤␥ was
examined by adding increasing concentrations of G␤␥ to the bath while
recording the channel activity. Patches were excised into the standard
bath solution, and basal activity was recorded for 2.5 min before G␤␥
was added to an initial concentration of 2 nM. Additional G␤␥ was added
incrementally at 2.5-min intervals until a final concentration of 60 or 90
nM was reached. 2.5 min after the highest G␤␥ concentration was
attained, 100 ␮M GTP␥S was added to the bath. NPo was determined for
the final minute of each G␤␥ concentration step. NPo values were
normalized to the final activity level 1.5 min after the addition of
GTP␥S for each patch in order to derive relative NPo values. Relative
NPo values from all patches were averaged, and the dose-response
curve was determined as the best fit to the mean data.
Western and Coimmunoprecipitation Assays Using Recombinant
GIRK2/GIRK3—Membrane proteins were isolated from single 100mm2 dishes of CHO-K1 cells transfected with GIRK1 plus GIRK2,
GIRK1 plus GIRK3, and GIRK2 plus GIRK3. To serve as a control,
membrane proteins were also isolated from untransfected cells. The
cells were washed twice with PBS before being lifted from the dish with
5 mM EDTA in PBS. The cells were pelleted by centrifugation at 1200 ⫻
g for 5 min. Cold hypotonic 5/5/5 lysis buffer (5 mM each of Tris-HCl,
EDTA, and EGTA; pH 8) with protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 2 ␮g/ml each of aprotinin, pepstatin, and leupeptin) was added to the cells, which were lysed on ice by drawing them
through a 23-gauge needle 10 times and a 27-gauge needle four times.
Crude membranes were obtained by centrifugation at 120,000 ⫻ g for
15 min at 4 °C. The crude membranes were resuspended in 5/5/5 buffer
with protease inhibitors, and the proteins were precipitated with 100%
(w/v) trichloroacetic acid (Sigma). The precipitated proteins were dissolved in 1⫻ SDS sample buffer and assayed by Western blot with
anti-GIRK1 fusion protein antibody ␣CSh (anti-CSh serum, generated
against the carboxyl-terminal 156 amino acids of GIRK1) or with antiGIRK1 antibody (Alomone Laboratories, Jerusalem, Israel).
Epitope-tagged GIRK2-Myc and GIRK3-AU1 constructs were transfected in the recombinant coimmunoprecipitation experiments. The
GIRK2-Myc construct has been described previously (20). The AU1
epitope, DTYRYI, was inserted after the initiator methionine in the
pcDNA3.1-GIRK3 plasmid using the QuickChangeTM mutagenesis kit
(Stratagene, La Jolla, CA). The primer sequences used were 5⬘-CCCGCTGCGGCCGCCATGGACACTTACCGCTATATTGCGCAGGAGAACG-3⬘ and 5⬘-GCGTTCTCCTGCGCAATATAGCGGTAAGTGTCCATGGCGGCCGCAGCGG-3⬘. Positive clones were identified by the
introduction of a unique FspI restriction site, and the integrity of the
tagged GIRK3 sequence was verified by DNA sequencing.
COS-7 cells were used for the recombinant coimmunoprecipitation
experiments because they produced significantly more expressed protein than the CHO-K1 cells. COS-7 cells have been used to examine
GIRK channels in previous functional studies (7, 8, 10, 17, 22, 23) and
are known to produce functional GIRK channels. Single 100-mm2
dishes of COS-7 cells expressing GIRK2-Myc and GIRK3-AU1 were
washed twice with PBS (22 °C) before being lifted from the dish with 5
mM EDTA in PBS. The cells were pelleted by centrifugation at 1200 ⫻
g for 5 min. Hypotonic 5/5/5 lysis buffer with protease inhibitors was
added to the cells, which were lysed on ice by drawing them through a
23-gauge needle 10 times. The lysed cells were centrifuged at 150,000 ⫻
g for 15 min at 4 °C to obtain a crude particulate fraction. This fraction
was resuspended in 5/5/5 with protease inhibitors and centrifuged
through a 20% sucrose cushion at 150,000 ⫻ g for 35 min at 4 °C. The
resulting pellet was solubilized in 1% CHAPS, 10 mM HEPES, 300 mM
NaCl, 5 mM EGTA, and 5 mM EDTA with protease inhibitors at pH 8.0.
For immunoprecipitation, samples were precleared with Gammabind-G Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C.
Centrifuged ascites fluid for AU1 monoclonal antibodies (BabCO, Richmond, CA) was used at a 1:200 dilution. Samples were incubated for 4 h
at 4 °C on a rotator. Bound proteins were eluted with 1⫻ SDS sample
buffer at 55 °C for 20 min. The eluted proteins were analyzed by
Western blot with anti-GIRK2 antibody (Alomone Laboratories) and
rabbit anti-GIRK3 antibody C-312 (Cocalico Biologicals, Inc., Reamstown,
PA) against a carboxyl-terminal peptide antigen (SIPSRLDEKVEEEGAGEGGR). The specificity of anti-GIRK3 antibody C-312 was confirmed
against a panel of recombinant GIRK proteins expressed in COS-7 cells.
Isolation and Solubilization of Mouse Brain Membranes—Whole
brains were removed from wild-type and GIRK3 knockout mice.2 The
tissue was minced in homogenization buffer (320 mM sucrose, 4 mM
HEPES-NaOH, pH 7.4) with protease inhibitors (50 ␮g/ml leupeptin,
100 ␮g/ml phenylmethysulfonyl fluoride, 1 ␮g/ml aprotinin, and 2 ␮g/ml
pepstatin) on ice before homogenization with a Polytron homogenizer
(2 ⫻ 15 s). The homogenized tissue was centrifuged for 10 min at 1000 ⫻
g to remove nuclei and large cellular fragments. The supernatant was
centrifuged for 30 min at 120,000 ⫻ g at 4 °C to pellet the membranes.
The pelleted membranes were resuspended in immunoprecipitation
buffer (10 mM HEPES, 100 mM NaCl, 5 mM EDTA, pH 7.53) with 1%
Triton X-100 (Sigma). Protein was quantified using the Bio-Rad Protein
Assay (Bio-Rad). Solubilized membranes were stored at ⫺80 °C.
Immunoprecipitation of GIRK Complexes from Mouse Brain—
GIRK2/GIRK3 complexes were purified by serial immunoprecipitation.
Any potential GIRK1-containing complexes were eliminated in the first
two rounds of immunoprecipitation so that any complex containing
GIRK1, GIRK2, and GIRK3 would not be mistakenly identified as one
containing only GIRK2 and GIRK3. The third round was designed to
immunoprecipitate remaining complexes that contained only GIRK2
and/or GIRK3 subunits.
Each serial experiment tested three wild-type samples against three
GIRK3 knockout mouse samples. One tube in each group was a control
containing no antibody. Anti-GIRK1 serum ␣CSh was prebound for 8 h
to protein A-Sepharose beads (Amersham Pharmacia Biotech) at a 1:92
dilution. The resin was washed 7⫻ with 1-ml aliquots of immunoprecipitation buffer before 200 ␮g of whole brain membrane proteins from
wild-type or GIRK3 knockout mice were added. Samples were incubated at 4 °C overnight on a rotator and centrifuged, and the supernatants were transferred to fresh tubes containing additional ␣CSh bound
to protein A-Sepharose. For the second round of GIRK1 removal, the
samples were incubated at 4 °C for 3 h on a rotator. Afterward, supernatant samples were assayed for GIRK1 by Western blotting with
␣CSh. Supernatants were transferred to fresh tubes containing antiGIRK2 antibody (Alomone Laboratories) at a 1:174 dilution or antiGIRK3 antibody C-312 at a 1:67 dilution. The antibodies were preincubated with protein A-Sepharose and washed seven times with 1-ml
aliquots of immunoprecipitation buffer prior to sample addition and
incubation at 4 °C overnight. Supernatants were removed before the
resin was washed, and bound proteins were eluted with 1⫻ SDS sample
buffer with ␤-mercaptoethanol (Sigma; 55 °C for 20 min). Eluted proteins were run on 10% Bis-Tris NuPAGE gels in MOPS buffer (Novex,
San Diego, CA), providing maximal separation of proteins in the 35–
55-kDa range. To avoid confusing signals from the GIRK proteins with
the heavy chains of the immunoglobulins used to purify them, the blots
were probed with biotinylated anti-GIRK2 (Alomone Laboratories) and
biotinylated C-312 anti-GIRK3 antibodies, followed by incubation with
strepavidin-horseradish peroxidase. The antibodies were biotinylated
using E-Z-Link NHS-LC-Biotin (Pierce).
RESULTS
Cotransfection of GIRK2 and GIRK3 Yields a Functional
Channel in CHO-K1 Cells—In order to examine the properties
of a potential channel composed of GIRK2 and GIRK3 subunits, CHO-K1 cells were cotransfected with GIRK2 and
GIRK3 and assayed for channel activity by patch clamp. Insideout patches of transfected cells contained channels that could
be activated by application of GTP␥S to the cytoplasmic face of
the patch and which displayed inward rectification (Fig. 1). The
slope conductance of the channel was 31 ⫾ 1.7 picosiemens (⫾
S.E.; n ⫽ 14), smaller than that observed for GIRK1/GIRK2,
GIRK1/GIRK3, and GIRK1/GIRK4 channels (35, 39, and 37
picosiemens, respectively) in our previous study (1) but still
within the range of conductance values reported for GIRK
channels (24). The ␶o at ⫺100 mV for the GIRK2/GIRK3 chan2
Tanya M. Jelacic, Matthew E. Kennedy, Kevin Wickman, and David
E. Clapham, manuscript in preparation.
GIRK2/GIRK3 Channels
36213
FIG. 2. Dose-response curve of GIRK2/GIRK3 channels to G␤␥.
Increasing amounts of G␤␥ were applied to the cytoplasmic face of
inside-out patches expressing GIRK2/GIRK3 at set time intervals (see
“Experimental Procedures”). At the end of each experiment, 100 ␮M
GTP␥S was applied. The activity level for each concentration of G␤␥
was quantitated as NPo and normalized to the NPo for the GTP␥S step
for each patch to derive relative NPo values. Relative NPo values were
plotted against log[G␤␥], and a sigmoidal curve was fit to the mean
data. Channel activity increased in response to increasing G␤␥, with a
half-maximal concentration of 53 nM (n ⫽ 4 patches). GIRK1/GIRK3
data are replotted from Ref. 1 for comparison.
FIG. 1. Inwardly rectifying channel currents are seen when
GIRK2 and GIRK3 are co-expressed in CHO-K1 cells. CHO-K1
cells expressing GIRK2 and GIRK3 were analyzed by patch clamp in
the inside-out configuration in symmetrical 140 mM K⫹. Channels were
activated by application of 40 ␮M GTP␥S to the cytoplasmic face of the
patch. A, records were made at transmembrane potentials both positive
and negative to the reversal potential of 0 mV to verify inward rectification. B, channel currents were recorded at various transmembrane
potentials. Current-voltage relation reveals a unitary conductance of 31
picosiemens (pS) (mean ⫾ S.E.; n ⫽ 14 patches). C, open time distribution at ⫺100 mV is shown with a dwell time histogram.
nel was 1.3 ms (n ⫽ 11), similar to that for GIRK1/GIRK2,
GIRK1/GIRK3, and GIRK1/GIRK4 channels at ⫺80 mV (1.0,
1.1, and 1.2 ms, respectively) (1). These results were surprising, considering that any one of the GIRK2, GIRK3, or GIRK4
subunits expressed alone yields spiky, irregular channels with
very brief open times (1, 3– 6).
Since activation by G␤␥ is a hallmark feature of GIRK channels, the concentration dependence of GIRK2/GIRK3 channel
activation was determined. Inside-out patches containing the
channel were exposed to increasing concentrations of G␤␥ at
regularly timed intervals until a final concentration was
reached (see Ref. 25). At a set time interval after reaching the
final G␤␥ concentration, a saturating concentration of GTP␥S
was added to the bath. The channel activity, quantitated as
NPo at each concentration of G␤␥, was determined and normalized to the NPo after the addition of GTP␥S in order to derive
relative values. The relative activity was plotted against log
concentrations of G␤␥ to determine the concentration required
to elicit half-maximal activation of the channel (Fig. 2). While
GIRK1/GIRK4 and GIRK1/GIRK3 are activated by G␤␥ with a
half-maximal concentration of 11 nM (1, 23), the GIRK2/GIRK3
channel displays a half-maximal concentration of 53 nM, a
4.8-fold lower sensitivity to G␤␥. Two-way factorial analysis of
variance revealed no significant difference in the slopes of the
FIG. 3. Cotransfection of GIRK2 and GIRK3 does not induce
expression of GIRK1 in CHO-K1 cells. CHO-K1 cells were co-transfected with GIRK1 plus GIRK2, GIRK1 plus GIRK3, or GIRK2 plus
GIRK3. Control cells were not transfected. Membrane proteins were
harvested from all cells and analyzed for GIRK1 by Western blot. Only
cells transfected with the GIRK1 construct expressed GIRK1 protein.
two dose-response curves, but GIRK1/GIRK3 is significantly
more sensitive to G␤␥ than GIRK2/GIRK3; F (1, 25) ⫽ 8.77, p ⫽
0.007.
GIRK1 Is Not Expressed in CHO-K1 Cells Cotransfected with
GIRK2 and GIRK3—CHO-K1 cells do not normally express
GIRK channel subunits. To rule out the possibility that cotransfection of GIRK2 and GIRK3 could induce the cells to
express GIRK1, membrane proteins were harvested from cotransfected cells and tested for the presence of GIRK1 protein
by Western blotting. CHO-K1 cells transfected with GIRK1/
GIRK2 and GIRK1/GIRK3 were used as positive controls for
GIRK1 expression, while untransfected CHO-K1 cells were
used as a negative control. Two separate anti-GIRK1 antibodies were used to test for GIRK1 expression. In both cases,
36214
GIRK2/GIRK3 Channels
FIG. 4. GIRK2 coimmunoprecipitates with GIRK3 after co-expression in COS-7 cells. A, Western blot showing specific recognition of
GIRK3 by anti-GIRK3 antibody C-312 from among a panel of recombinant GIRKs expressed in COS-7 cells. B, Western blot showing specific
recognition of GIRK2 by anti-GIRK2 antibody from among a panel of recombinant GIRKs expressed in COS-7 cells. C, GIRK2-Myc and GIRK3-AU1
were cotransfected in COS-7 cells. Membrane proteins were harvested and subjected to an immunoprecipitation with ascites fluid for monoclonal
AU1 antibodies. On the left, a Western blot probed with anti-GIRK3 antibody demonstrated the presence of GIRK3 in the AU1 immunoprecipitate.
On the right, reprobing the same blot with the anti-GIRK2 antibody after stripping demonstrates that GIRK2 coimmunoprecipitated with GIRK3.
GIRK1 protein could be detected only in cells that had been
transfected with GIRK1 cDNA (Fig. 3).
GIRK2 and GIRK3 Form a Complex in Transfected Cells—To
examine whether or not cotransfected GIRK2 and GIRK3 subunits were able to form heteromultimeric complexes, we coimmunoprecipitated the GIRK channel proteins from COS-7 cells
transfected with epitope-tagged GIRK subunits. After cotransfection with the GIRK2-Myc and GIRK3-AU1 constructs, membrane proteins from COS-7 cells were harvested, and ascites
fluid for monoclonal AU1 antibodies was used to immunoprecipitate GIRK3-AU1 and any associated proteins. These proteins were eluted and transferred to a membrane for Western
blot analysis with subunit-specific anti-GIRK2 and anti-GIRK3
antibodies. The specific recognition of their respective GIRK
proteins by these antibodies can be seen in Fig. 4, A and B.
Analysis of the eluted proteins by Western blot demonstrated
the presence of both GIRK3 and GIRK2 in the AU1 ascites
pellet (Fig. 4C). These coimmunoprecipitation data strongly
support the notion that GIRK2 and GIRK3 subunits can combine to form a heteromultimeric complex in transfected mammalian cells.
GIRK2/GIRK3 Complexes Are Present in Native Brain—To
test whether heteromultimeric complexes of GIRK2 and GIRK3
subunits might exist in vivo, we performed coimmunoprecipitation experiments using mouse brain as the tissue source.
Membranes solubilized in 1% Triton X-100 buffer were prepared from wild-type and GIRK3 knockout (control) mouse
brains.2 Unlike the heterologous expression system, the native
system is complicated by the presence of GIRK1. A straightforward GIRK2/GIRK3 coimmunoprecipitation experiment would
have ambiguous results because it would be uncertain whether
or not the complex containing GIRK2 and GIRK3 also contained GIRK1. To avoid this ambiguity, the solubilized membranes were first immunodepleted of GIRK1 by two rounds of
GIRK1 immunoprecipitation before immunoprecipitation of
GIRK2 and GIRK3.
After the second round of GIRK1 immunodepletion, small
supernatant samples were tested for GIRK1 by Western analysis. As Fig. 5A shows, GIRK1 protein was successfully removed, leaving a GIRK1-free protein pool from which we could
immunoprecipitate GIRK2 and GIRK3. The final immunoprecipitates and control supernatants were analyzed by Western
blot for GIRK2 and GIRK3 coimmunoprecipitation.
Fig. 5B shows the final immunoprecipitates and control su-
GIRK2/GIRK3 Channels
pernatants probed with anti-GIRK2 antibody. The GIRK2 signal from native brain membranes consistently appeared as a
doublet, suggesting that at least two GIRK2 isoforms are present (26, 27). GIRK2 protein was recovered from the control
supernatant and both the anti-GIRK2 and anti-GIRK3 immunoprecipitates of the wild-type samples but was recovered only
from the control supernatant and the anti-GIRK2 immunoprecipitate of the GIRK3 knockout samples. The presence of
GIRK2 in the anti-GIRK3 wild-type immunoprecipitate together with its absence from the anti-GIRK3 knockout immunoprecipitate indicates that GIRK2 was pulled down by the
anti-GIRK3 antibody most likely because it was complexed
with GIRK3. Fig. 5C shows the final immunoprecipitates and
control supernatants probed with an anti-GIRK3 antibody.
GIRK3 was recovered from the control supernatant and both
the anti-GIRK2 and anti-GIRK3 immunoprecipitates of the
wild-type samples. As expected, no GIRK3 signal was seen for
the GIRK3 knockout samples. The presence of GIRK3 in the
anti-GIRK2 wild-type immunoprecipitate reinforces the evidence of coimmunoprecipitation seen with the anti-GIRK3 antibody. These results demonstrate that complexes containing
GIRK2 and GIRK3 without GIRK1 can be immunoprecipitated
from native brain.
DISCUSSION
We have demonstrated that GIRK2/GIRK3 channels are
functional, exist in native brain tissue, and are less sensitive to
G␤␥ than GIRK1-containing channels. When GIRK2, GIRK3,
or GIRK4 subunits are transfected alone, each gives rise to
“spiky” channels with very brief open times (⬍0.5 ms) and
variable single channel conductance (20 – 40 picosiemens) (1, 3,
5, 6). Co-transfecting either subunit with GIRK1 yields a heteromultimeric channel with characteristics like the prototypical GIRK channel, IKACh. The uniqueness of GIRK1 has attracted a series of hypotheses to account for its long carboxylterminal tail, including the idea that it modifies the gating of
the GIRK heteromultimer to yield longer open times. As we
have shown, co-transfecting GIRK2 and GIRK3 into CHO cells
verifiably lacking GIRK1 gives rise to an inwardly rectifying
current with single channel properties much like those described for the GIRK1-containing channels. It thus appears
that the longer open time does not depend on the presence of
GIRK1 but that heteromultimerization places the subunits in
the proper conformation to achieve a longer open time and
quantized conductance level.
Despite its similarities with respect to open time and conductance, the GIRK2/GIRK3 channel differed from the GIRK1/
GIRKx channels in being nearly 5-fold less sensitive to activation by G␤␥. A recent study has shown that residues proximal
to the membrane in the carboxy tail region of GIRK4 are
important for binding by G␤␥ and critical for activation of
IKACh (10). The similarity of this region (e.g. C216) in GIRK2
and GIRK3 probably accounts in part for the similar responses
of the GIRK1/GIRK4 and GIRK1/GIRK3 channels to G␤␥ (1).
However, the same study and others have demonstrated that
GIRK1 also binds G␤␥ (11, 12, 28). Thus, G␤␥ binding to
GIRK1 may have a potentiating effect, or perhaps GIRK1 and
GIRK4 both contribute to the binding site for G␤␥, and
GIRK1’s contribution may be to increase the affinity for G␤␥.
Alternatively, GIRK1 may contribute to or affect the binding
site for a modulating molecule such as inositol 1,4,5-bisphosphate, which has been shown to play an important role in the
activation of IKACh by G␤␥ and by intracellular Na⫹ (29 –33).
The lower sensitivity of GIRK2/GIRK3 channels to activation by G␤␥ may partly explain why the GIRK currents in
Xenopus oocytes co-injected with cRNAs for these subunits are
so small (16 –19). Additionally, GIRK3 apparently expresses at
36215
FIG. 5. Purification of GIRK2/GIRK3 complexes from native
brain tissue by serial immunoprecipitation. A, after two rounds of
immunodepletion (see “Experimental Procedures”), GIRK1 protein had
been effectively removed. B, the GIRK1-free samples of membrane
proteins were immunoprecipitated with anti-GIRK2 or anti-GIRK3 antibodies (Ab) to yield the final immunoprecipitates. Among the wild type
(WT) samples, GIRK2 was recovered from the control supernatant (sup)
and both the anti-GIRK2 and anti-GIRK3 immunoprecipitates (ppt).
Among the GIRK3 knockout samples, GIRK2 was recovered only from
the control supernatant and the anti-GIRK2 immunoprecipitate. C,
among the wild type samples, GIRK3 was recovered from the control
supernatant and both the anti-GIRK2 and anti-GIRK3 immunoprecipitates. Among the GIRK3 knockout samples, no GIRK3 signal was
detected as expected.
low levels in oocytes compared with the other GIRK subunits
(16 –19, 34). The combination of poor expression of the channel
with its decreased sensitivity to G␤␥ may account for the small
peak currents observed for GIRK2/GIRK3 in Xenopus oocytes.
In contrast, GIRK3 and GIRK2/GIRK3 express well in mammalian cells.
In addition to providing enhanced G␤␥ sensitivity, GIRK1
may also provide a second localization mechanism via its
unique carboxy tail. Additional diversity comes from the fact
that GIRK3 and the long form of GIRK2 both have potential
PDZ-binding motifs at their carboxyl termini. The variety of
GIRK channel combinations may allow for specificity of localization of channels in the neuron, at pre- or postsynaptic sites
for example, or for linking of channel subtypes to specific neurotransmitter receptors.
Most regions in the brain have the potential to produce
GIRK2/GIRK3 channels because they express GIRK1, GIRK2,
and GIRK3 mRNAs (13–15). The expression of GIRK1 does not
preclude the formation of complexes that do not contain
GIRK1, and cells may produce more than one type of GIRK
channel. This is clearly illustrated by the presence of GIRK4
homomeric complexes in atrial cells expressing IKACh (35). It is
possible that a neuron that produces GIRK1/GIRK2 and
GIRK1/GIRK3 channels may also produce GIRK2/GIRK3
channels. GIRK2/GIRK3 channels have a different sensitivity
to activation by G␤␥ and may be localized differently than
channels containing GIRK1. Thus, there may be regions within
neurons where GIRK2/GIRK3 channels are more common than
GIRK1/GIRK2 or GIRK1/GIRK3 channel complexes.
In summary, we have demonstrated the existence in brain of
a heteromultimeric GIRK channel composed of GIRK2 and
GIRK3 subunits. These channels have a similar open time to
the GIRK1/GIRKx channels but have a slightly smaller conductance and less sensitivity to activation by G␤␥. The discovery of this channel, with its lesser sensitivity to G␤␥ and hence
36216
GIRK2/GIRK3 Channels
lower threshold of activation, along with its potential for differential localization from the GIRK1/GIRKx channels, broadens the functional scope of the GIRK family of channels.
Acknowledgments—We thank Grigory Krapivinsky for providing purified bovine brain G␤␥ and David Zurakowski for help with the statistics. We also thank Christov Roberson and Stephen Sims for critical
reading of the manuscript.
REFERENCES
1. Jelacic, T. M., Sims, S. M., and Clapham, D. E. (1999) J. Membr. Biol. 169,
123–129
2. Krapivinsky, G., Krapivinsky, L., Velimirovic, B., Wickman, K., Navarro, B.,
and Clapham, D. E. (1995) J. Biol. Chem. 270, 28777–28779
3. Velimirovic, B. M., Gordon, E. A., Lim, N. F., Navarro, B., and Clapham, D. E.
(1996) FEBS Lett. 379, 31–37
4. Krapivinsky, G., Gordon, E., Wickman, K., Velimirovic, B., Krapivinsky, L.,
and Clapham, D. E. (1995) Nature 374, 135–141
5. Chan, K. W., Sui, J. L., Vivadou, M., and Logothetis, D. E. (1997) J. Biol.
Chem. 272, 6548 – 6555
6. Vivadou, M., Chan, K. W., Sui, J., Jan, L. Y., Reuveny, E., and Logothetis, D. E.
(1997) J. Biol. Chem. 272, 31553–31560
7. Kennedy, M. E., Nemec, J., and Clapham, D. E. (1996) Neuropharmacology 35,
831– 839
8. Kennedy, M. E., Nemec, J., Corey, S., Wickman, K., and Clapham, D. E. (1999)
J. Biol. Chem. 274, 2571–2582
9. Chan, K. W., Sui, J. L., Vivaudou, M., and Logothetis, D. E. (1996) Proc. Natl.
Acad. Sci. U. S. A. 93, 14193–14198
10. Krapivinsky, G., Kennedy, M., Nemec, J., Medina, I., Krapivinsky, L., and
Clapham, D. (1998) J. Biol. Chem. 273, 16946 –16952
11. Huang, C.-L., Jan, Y. N., and Jan, L. Y. (1997) FEBS Lett. 405, 291–298
12. He, C., Zhang, H., Mirshahi, T., and Logothetis, D. E. (1999) J. Biol. Chem.
274, 12517–12524
13. Chen, S. C., Ehrhard, P., Goldowitz, D., and Smeyne, R. J. (1997) Brain Res.
778, 251–264
14. Karschin, C., Dissmann, E., Stuhmer, W., and Karschin, A. (1996) J. Neurosci.
16, 3559 –3570
15. Kobayashi, T., Ikeda, K., Ichikawa, T., Abe, S., Togashi, S., and Kumanishi, T.
(1995) Biochem. Cell Biol. 208, 1166 –1173
16. Schoots, O., Wilson, J. M., Ethier, N., Bigras, E., Hebert, T. E., and Van Tol,
H. H. M. (1999) Cell. Signal. 11, 871– 883
17. Wischmeyer, E., Doring, F., Wischmeyer, E., Spauschus, A., Thomzig, A., Veh,
R., and Karschin, A. (1997) Mol. Cell. Neurosci. 9, 194 –206
18. Lesage, F., Guillemare, E., Fink, M., Duprat, F., Heurteaux, C., Fosset, M.,
Romey, G., Barhanin, J., and Lazdunski, M. (1995) J. Biol. Chem. 270,
28660 –28667
19. Kofuji, P., Davidson, N., and Lester, H. A. (1995) Proc. Natl. Acad. Sci. U. S. A.
92, 6542– 6546
20. Navarro, B., Kennedy, M. E., Velimirovic, B., Bhat, D., Peterson, A., and
Clapham, D. E. (1996) Science 272, 1950 –1953
21. Sigworth, F. J., and Sine, S. M. (1987) Biophys. J. 52, 1047–1054
22. Dissmann, E., Wischmeyer, E., Spauschus, A., Von, P. D., Karschin, C., and
Karschin, A. (1996) Biochem. Cell Biol. 223, 474 – 479
23. Spauschus, A., Lentes, K.-U., Wischmeyer, E., Dissmann, E., Karschin, C., and
Karschin, A. (1996) J. Neurosci. 16, 930 –938
24. Dascal, N. (1997) Cell. Signal. 9, 551–573
25. Wickman, K. D., Iniguez-Lluhl, J. A., Davenport, P. A., Taussig, R., Krapivinsky, G. B., Linder, M. E., Gilman, A. G., and Clapham, D. E. (1994) Nature
368, 255–257
26. Wei, J., Hodes, M. E., Piva, R., Feng, Y., Wang, Y., Ghetti, B., and Dlouhy, S. R.
(1998) Genomics 51, 379 –390
27. Isomoto, S., Kondo, C., Takahashi, N., Matsumoto, S., Yamada, M., Takumi,
T., Horio, Y., and Kurachi, Y. (1996) Biochem. Cell Biol. 218, 286 –291
28. Kunkel, M. T., and Peralta, E. G. (1995) Cell 83, 443– 449
29. Sui, J. L., Petit-Jacques, J., and Logothetis, D. E. (1998) Proc. Natl. Acad. Sci.
U. S. A. 95, 1307–1312
30. Petit-Jacques, J., Sui, J. L., and Logothetis, D. E. (1999) J. Gen. Physiol. 114,
673– 684
31. Huang, C.-L., Feng, S., and Hilgemann, D. W. (1998) Nature 391, 803– 806
32. Ho, I. H. M., and Murrell-Lagnado, R. D. (1999) J. Biol. Chem. 274, 8639 – 8648
33. Ho, I. H. M., and Murrell-Lagnado, R. D. (1999) J. Physiol. 520, 645– 651
34. Lesage, F., Duprat, F., Fink, M., Guillemare, E., Coppola, T., Lazdunski, M.,
and Hugnot, J. P. (1994) FEBS Lett. 353, 37– 42
35. Corey, S., and Clapham, D. E. (1998) J. Biol. Chem. 273, 27499 –27504