From the Howard Hughes Medical Institute, Children's
Hospital, Boston, Massachusetts 02115, ¶ Mayo Foundation,
Rochester, Minnesota 55905, and the § Department of
Cardiology, Children's Hospital, Harvard Medical School,
Boston, Massachusetts 02115
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ABSTRACT |
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GIRK1 and GIRK4 subunits combine to form the
heterotetrameric acetylcholine-activated potassium current
(IKACh) channel in pacemaker cells of the heart. The
channel is activated by direct binding of G-protein G Physiological changes in a cell membrane's permeability to
potassium (K+) ions are in large part mediated by integral
membrane K+-selective channels. Alteration of
K+ flux is a critical mechanism for controlling cell
excitability in the heart, nervous system, and exocrine tissues. The
inward rectifier K+ channels
(IRKs)1 contain two
transmembrane domains in contrast to the six transmembrane domains of
their voltage-gated K+ channel relatives (see Refs. 1-3
for review). Like the Shaker family of K+ channels, IRKs
have the characteristic K+ ion selectivity filter sequence
(GYG) in their pore domain. Inward rectifiers conduct K+
ions inwardly better than in the outward direction due to intracellular block of the channel pore by Mg2+ ions (4) and polyamines
(5). IKACh is a unique inwardly rectifying potassium
current activated by hormones and neurotransmitters such as
acetylcholine (ACh), somatostatin, and adenosine, via stimulation of
G-protein-coupled receptors (6). ACh released from the vagus nerve
binds and activates muscarinic M2 receptors located on
cardiac pacemaker cells. M2 receptors selectively couple to
the pertussis toxin-sensitive class (Gi/Go) of
heterotrimeric G-proteins composed of The cloning of GIRK1, the first identified GIRK
(G-protein-activated inwardly
rectifying K+ channel) subunit,
represented the first step toward characterizing native
IKACh (9, 10). The GIRK1 cDNA was believed to represent native atrial IKACh, since oocytes co-injected with GIRK1
and muscarinic M2 receptor cRNAs displayed
ACh-dependent inwardly rectifying K+ currents
(9, 10). However, biochemical purification of GIRK1 subunits from
native cardiac atrial tissue revealed that GIRK1 subunits physically
associate with a closely related inward rectifier K+
channel subunit, GIRK4, to form the native cardiac IKACh
channel (11). Further studies revealed that native IKACh
exists as a heterotetramer composed of two GIRK1 and two GIRK4 subunits
(12).
G The GIRK family of inward rectifier K+ channels contains
two additional members, GIRK2 and GIRK3 (23, 24), which, along with
GIRK1 and GIRK4, are differentially expressed in the brain (25).
Alternative splicing of the GIRK2 gene results in at least four distinct GIRK2 mRNAs, GIRK2-1, GIRK2A, GIRK2B, and
GIRK2C, which differ in the length and amino acid sequence of amino and carboxyl domains (24, 26). Coexpressed GIRK2-1 and GIRK1 subunits produce IKACh-like channels (27, 28). A point mutation (GYG Coexpression of GIRK1 and GIRK4 subunits in Xenopus oocyte
or mammalian cell expression systems such as Chinese hamster ovary (CHO) or COS-7 cells results in
G We previously showed that GIRK1 and GIRK4 subunits display dramatically
different localization patterns in permeabilized COS-7 cells with GIRK4
being localized to the cell surface, whereas GIRK1 localized to the
cytoskeleton (35). Using an extracellular epitope tag on GIRK1, we
demonstrate that GIRK1 requires association with GIRK4 for cell surface
localization. GIRK4 associates with GIRK1 either during or shortly
after subunit synthesis and allows appropriate glycosylation of GIRK1
subunits to a form seen in native atrial tissue. We created chimeras of
GIRK1 and GIRK4 subunits that localize to the cell surface to identify
potential domains involved in the differential localization of the
subunits. Specifically, chimeras containing the C-terminal tail of
GIRK1 were retained inside the cell, while chimeras containing the C
terminus of GIRK4 were expressed at the cell surface. Using deletion
analysis of GIRK4, we narrowed down the region of GIRK4 required for
cell surface localization of GIRK1/GIRK4 heterotetramers to amino acids 375-399. The importance of GIRK4's role in cell surface localization and processing of heterotetrameric IKACh is underscored by
the absence of mature glycosylation and the intracellular localization of GIRK1 in dissociated atrial myocytes from GIRK4 knockout mice.
Cell Culture and Transfections--
COS-7 cells were cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum at 37 °C, 5%
CO2. Cells were plated at 2 × 106
cells/100-mm dish 1 day prior to transfection. COS-7 cells were transfected using either Lipofectamine (Life Technologies) or TransIT
LT-1 (PanVera Corp., Madison, WI). CHO cells were cultured in Ham's
F-12 medium (Life Technologies) supplemented with 10% fetal calf
serum. CHO cells were plated at 40% confluence onto glass coverslips
and transfected with channel cDNAs and tracer amounts of
pGREEN-Lantern (Life Technologies) as a marker to identify transfected cells.
Mutagenesis--
Construction and characterization of the
C-terminally tagged rat GIRK1-AU5 and rat GIRK4-AU1 epitope-tagged
subunits has been described (35). The Flag epitope sequence (DYKDDDDK)
was introduced into the putative extracellular region of the GIRK1-AU5
cDNA by oligonucleotide-directed mutagenesis using the site
elimination method (Transformer Mutagenesis;
CLONTECH, Palo Alto, CA) between amino acids 114 and 115 of GIRK1 to create the GIRK1-Flag cDNA. To eliminate GIRK1
N-linked glycosylation, the N119D mutation was introduced
into the GIRK1-Flag to produce the GIRK1(N119D)-Flag cDNA.
GIRK1(N119D)-Flag subunits displayed similar functional properties as
wild type GIRK1 subunits when coexpressed with the GIRK4 subunit (data
not shown). Truncation of GIRK1 at amino acid 373 was performed by
introduction of the 6-amino acid epitope AU5 (TDFYLK) followed by a TGA
stop codon and a unique XbaI site by polymerase chain
reaction (PCR) using Vent polymerase (New England Biolabs, Beverly,
MA). A 332-base pair PmlI/XbaI fragment derived
from the PCR product was subcloned into the wild type-containing pCDNA3-GIRK1 vector and sequenced. The GIRK4:GIRK1-(373-501)
chimera was created by introduction of a unique NotI site at
the C terminus of GIRK4 and at amino acid 373 of GIRK1. A 417-base pair
NotI/ApaI fragment from GIRK1 was subcloned into the
NotI/ApaI site of pCDNA3-GIRK4-AU1. A
cDNA encoding amino acids 373-501 followed by the Flag epitope was
created by PCR. The PCR-based strand overlap extension method was used
to create chimeras between GIRK1 and GIRK4 within the pore domain:
GIRK1-(1-141):GIRK4-(148-419), GIRK1:GIRK4-pore and GIRK4-(1-119):GIRK1-(114-501), GIRK4:GIRK1-pore. A second pair of
chimeras was generated just after the end of the second transmembrane domain: GIRK1-(1-194):GIRK4-(200-419), GIRK1-194:GIRK4-200 and GIRK4-(1-200):GIRK1-(194-501), GIRK4-200:GIRK1-194. The chimeric PCR products were TA-cloned using topoisomerase into the pCNDA3.1-TOPO vector (Invitrogen, Carlsbad, CA). GIRK4 deletion constructs were generated using the site elimination method described above by introducing the AU1 epitope followed by a stop codon after the indicated amino acid in Fig. 8. All constructs were verified by DNA sequencing.
Immunofluorescence Detection--
COS-7 cells were plated onto
100-mm dishes containing 25-mm coverslips prior to transfection.
Thirty-six to 48 h following transfection, the coverslips were
transferred to PBS supplemented with 0.5 mM each of
CaCl2 and MgCl2 (PBS C/M) and washed three times in PBS C/M at room temperature. The cells were fixed for 10 min
at room temperature with 4% paraformaldehyde in PBS followed by three
5-min washes with PBS. The cells were incubated in PBS supplemented
with Triton X-100 (0.2%) and 3% bovine serum albumin (0.2 µm
filtered; PBSTB) for 45 min to both permeabilize cells and block
nonspecific binding sites. Triton X-100 was eliminated from the PBSTB
solution in experiments designed to detect cell surface subunits
containing the extracellular Flag epitope. Primary and secondary
antibodies were diluted in PBSTB to the concentrations indicated in the
figure legends and incubated sequentially for 1 h at room
temperature. Following each incubation, the cells were washed four
times for 15 min each with 3 ml of PBSTB/coverslip. Mouse atrial
myocytes were isolated as described previously (8). Coverslips with
dissociated mouse atrial cells were processed as described above except
that the GIRK1-specific antifusion protein antibody anti-Csh (11) was
used at 2 µg/ml and a goat anti-rabbit rhodamine-conjugated secondary
antibody (Sigma) was used for detection. Coverslips were mounted onto
glass slides using Aqua-polymount (Polysciences Inc., Warrington, PA)
and viewed using a Zeiss LSM 410 laser scanning confocal microscope.
Electrophysiology--
Single channel recordings were performed
in the inside-out configuration, in symmetrical 140 mM
K+ solution (118.5 mM KCl, 5 mM
KOH/EGTA, 2 mM MgCl2, 10 mM
KOH/HEPES, pH 7.2, adjusted with concentrated HCl). The pipette
resistance was 2-5 megaohms. The holding potential was Assessment of GIRK1 Glycosylation--
Membranes from single
100-mm dishes of transiently transfected COS-7 cells were prepared as
described below. Endoglycosidase F or H (Boehringer Mannheim) treatment
was performed on equal amounts of membranes from COS-7 according to the
manufacturer's recommended procedure at 37 °C for 14 h.
Control samples not containing endoglycosidase enzymes were incubated
in parallel. The membrane fraction was isolated by centrifugation, and
the pellet was solubilized by incubation at 55 °C for 20 min in 1×
Laemmli sample buffer followed by SDS-PAGE and Western blotting with
GIRK1-specific polyclonal antibodies. Channel subunits were
immunoprecipitated using either the AU5 or AU1 antibody in experiments
assessing the effect of GIRK4-AU1 coexpression on the extent of mature
glycosylation of GIRK1. Proteins eluted from the Gammabind-G-Sepharose
were separated on a 11% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane using a semidry blotting apparatus for 40 min at 15 V. GIRK1 and GIRK4 proteins were identified by Western blotting with a
mixture of GIRK1 and GIRK4-specific affinity-purified polyclonal
antibodies using the enhanced chemiluminescence method for detection
(Amersham Pharmacia Biotech) Atrial membranes were prepared by first
manually dissecting away atria from three wild type or GIRK4 knockout
mice. Tissue was homogenized by three 30-s bursts of a Polytron
homogenizer in 100 mM NaCl, 20 mM imidazole, 1 mM dithiothreitol, pH 7.5. The homogenate was centrifuged
at 1000 × g for 10 min to pellet nuclei. The
supernatant was brought to 700 mM KCl and rotated for
1 h at 4 °C followed by centrifugation at 150,000 × g for 30 min. The membrane pellet was resuspended in PBS,
and protein levels were measured using the Bradford assay (Bio-Rad).
The equivalent of 200 µg of atrial membrane protein was
trichloroacetic acid-precipitated and solubilized in 1× SDS-sample buffer. Twenty-five µg of trichloroacetic acid-precipitated membrane protein from wild type or GIRK4 knockout mice was resolved on 10%
SDS-PAGE and then electroblotted to polyvinylidene difluoride followed
by Western blotting. Western blots were performed sequentially using
the anti-Csh (11), anti-GIRK1-(436-501) (Alamone Laboratories, Jerusalem, Israel), or KGAN2-216 directed against amino acids 1-22 of
GIRK1 (11). The GIRK4 antibody was raised against amino acids 404-418
in the C terminus. The GIRK4 antibody showed no cross-reactivity to
recombinant GIRK1, GIRK2-1, or GIRK3 (data not shown).
Immunoisolation of Epitope-tagged GIRKs from COS-7
Cells--
For pulse-labeling experiments, single 100-mm dishes of
COS-7 cells cotransfected with GIRK1-AU5 and GIRK4-AU1 subunits were methionine-deprived for 2 h followed by metabolic labeling with 50 µCi/ml of [35S]methionine (Amersham Pharmacia Biotech)
for 5, 15, 30, or 60 min. At each time point, the cells were cooled on
ice and washed twice with PBS and then lifted from the dish with PBS, 5 mM EDTA. The cells were pelleted by centrifugation at
1200 × g for 5 min at 4 °C. The cells were lysed on
ice by drawing the cell pellet through a 23-gauge needle 10 times in 5 mM Tris, 5 mM EDTA, 5 mM EGTA, pH
8.0 (5:5:5) supplemented with 10 µM phenylmethylsulfonyl fluoride and 2 µg/ml each of leupeptin, pepstatin, and aprotinin protease inhibitor mixture. A crude particulate fraction was obtained by centrifugation at 150,000 × g for 15 min at
4 °C. The resulting pellet was detergent-solubilized by drawing the
pellet through a 27-gauge needle 10 times in 750 µl of 1% CHAPS, 10 mM HEPES, 300 mM NaCl, 5 mM EGTA, 5 mM EDTA, pH 8.0, in aprotinin protease inhibitor mixture.
Insoluble material was removed by ultracentrifugation at 250,000 × g for 1 h. Samples were precleared for 1 h at
4 °C with 10 µl of Gammabind-G-Sepharose (Amersham Pharmacia
Biotech). Immunoprecipitations were performed at 4 °C for 4 h
by adding a 1:200 dilution of centrifuged ascites fluid for either the
AU1 or AU5 monoclonal antibodies (Babco, Richmond, CA) or a 10 µg/ml final concentration of M2 anti-Flag antibody (Sigma) and 10 µl of
Gammabind-G-Sepharose. The Gammabind-G-Sepharose was washed twice at
4 °C with 750 µl of solubilization buffer, twice with 750 µl of
solubilization buffer containing 0.2% CHAPS for 3 min each, and once
quickly with solubilization buffer containing 0.1% Triton X-100.
Gammabind-G-Sepharose-bound proteins were eluted with 1× nonreducing
Laemmli sample buffer at 55 °C for 20 min. For steady state labeling
experiments assessing the association of GIRK1( GIRK4 Subunits Are Required for Cell Surface Localization and
Mature Glycosylation of GIRK1 Subunits--
Expression of GIRK1
subunits alone in mammalian cells (CHO or COS-7) fails to produce
detectable IKACh-like channel activity in either whole-cell
recordings or inside-out patches, while GIRK4 subunits produce
G
Both GIRK1 and GIRK4 subunits contain N-linked glycosylation
consensus sites. Native atrial GIRK1 protein is represented by 55- and
~67-72-kDa bands on SDS-PAGE (11, 12). Enzymatic deglycosylation experiments demonstrated that the ~67-72-kDa GIRK1 band represented the glycosylated form of GIRK1, while the 55-kDa band was interpreted to be nonglycosylated GIRK1 (11). GIRK4 subunits are apparently not
glycosylated, despite the presence of a glycosylation motif (11). The
recombinant GIRK1-Flag protein migrates as a characteristic doublet
with apparent molecular masses of ~54 and ~56 kDa when expressed
alone in COS-7 cells. Two lines of evidence suggest that the upper band
of the GIRK1 doublet represents the core-glycosylated, immature form of
the GIRK1-Flag subunit. First, the intensity of the upper band of this
doublet is reduced by both endoglycosidase F and H treatment, whereas
the lower band was not (Fig.
2A). Densitometric scans of
each lane identified the shift from two closely associated peaks in the
control sample to a rightward shifted single peak in endoglycosidase F
and endoglycosidase H-treated membranes (Fig. 2A,
graph). Asparagine-linked glycosyl moieties can be
enzymatically removed from proteins using endoglycosidase F or H. However, endoglycosidase H requires that the glycosylated protein
not be modified by mannosiadase II, an enzyme present early in the
Golgi. Endoglycosidase F cleaves any form of N-linked sugar
moiety from a protein. The sensitivity of the upper band of the
GIRK1-Flag doublet to endoglycosidase F treatment suggests that it is a
glycosylated form of GIRK1, and the sensitivity to endoglycosidase H
treatment suggests that this form of GIRK1-Flag may reside in the
endoplasmic reticulum or early in the Golgi complex. Second, mutation
of asparagine 119 to aspartate (N119D) to eliminate the only
N-linked glycosylation consensus site resulted in a loss of
the upper band of the doublet. The resulting mutant GIRK1(N119D)-Flag
subunit co-migrated with the lower band of the wild type GIRK1-Flag
doublet (Fig. 2A). The ~73-kDa glycosylated form of
GIRK1-AU5 appears upon coexpression with GIRK4-AU1, (Fig.
2B). Coexpression of GIRK1-AU5 and GIRK4-AU1 subunits
followed by immunoprecipitation with the anti-AU1 antibody and Western
blotting for GIRK1 and GIRK4 subunits identified a ~73-kDa band not
found in either GIRK1- or GIRK4-only-expressing COS-7 cells. The
dependence of the mature glycosylated form of GIRK1-AU5 on GIRK4
coexpression suggests that the GIRK4 subunit promotes trafficking of
GIRK1 through the Golgi apparatus, where processing of the core
glycosylation sugar residues occurs (36, 37). Since the core sugar
residues common to all N-linked glycosylated proteins are
added to substrates in the endoplasmic reticulum, the core-glycosylated
GIRK1 subunits are likely to reside in a pre-Golgi or early Golgi
compartment. [35S]Methionine pulse-labeling experiments
were used to determine the point at which GIRK4 exerts its effects on
GIRK1 processing and cell surface localization. Coimmunoprecipitated
GIRK1-AU5/GIRK4-AU1 complexes were observed as soon as 5 min after
pulse-labeling COS-7 cells with [35S]methionine (Fig.
2C). This rapid association of GIRK1 and GIRK4 suggests that
the subunits interact either during or shortly after subunit
synthesis.
The Unique GIRK1 C-terminal Domain Is Required for Cytoskeletal
Localization of GIRK1 and Is Not Required for G
No channel activity was observed in inside-out patches from COS-7 cells
expressing the GIRK1( The C Terminus of GIRK4 Is Required for Cell Surface Localization,
whereas the C Terminus of GIRK1 Promotes Retention inside the
Cell--
Since deletions of the GIRK1 C terminus failed to produce
cell surface-localized GIRK1 subunits, we generated more extensive chimeras between the GIRK1-Flag and GIRK4-AU1 subunits to further study
the differential localization of GIRK1 versus GIRK4
subunits. One pair of chimeras generated in the pore region of the GIRK subunits contained either amino acids 1-141 of GIRK1 and 147-419 of
GIRK4 (GIRK1:GIRK4-pore) or amino acids 1-119 of GIRK4 and 114-501 of
GIRK1 (GIRK4:GIRK1-pore; see schematic in Fig.
6). Each construct contained the
extracellular Flag epitope. The GIRK1:GIRK4-pore chimera was detected
at the cell surface in nonpermeabilized COS-7 cells (Fig.
6C). Permeabilization of cells expressing the
GIRK1:GIRK4-pore chimera indicated that this chimeric subunit did not
significantly localize to the cytoskeleton (Fig. 6, compare
D and B). These data suggest that amino acids
1-141 are not sufficient for intracellular sequestration of GIRK1. The
reverse chimera, GIRK4:GIRK1-pore, was not detected at the cell surface
of nonpermeabilized cells (Fig. 6E). In permeabilized cells,
the GIRK4:GIRK1-pore chimera displayed a vesicular perinuclear staining
pattern, consistent with retention in the endoplasmic reticulum (Fig.
6F). Western blots of total membrane protein from cells
expressing wild type or chimeric subunits demonstrated that the
GIRK1:GIRK4-pore chimera expressed at levels comparable with that of
wild type GIRK1-Flag and GIRK4-AU1 (Fig.
7C, lane
3) and with the predicted molecular weight. However, the
GIRK4:GIRK1-pore chimera displayed significantly lower expression
levels that required a 60 times longer exposure to achieve a signal
similar to that of wild type subunits (Fig. 7C,
lanes 4 and 7).
We tested whether we could rescue the expression of the
GIRK4:GIRK1-pore chimera by extending the contribution of GIRK4
sequence in the GIRK4:GIRK1-pore chimera to include the second
transmembrane domain of GIRK4. The resulting GIRK4-200:GIRK1-194
chimera did display much higher expression levels similar to GIRK1-Flag
and GIRK4-AU1 and at the predicted molecular weight (Fig.
7C, lane 6). The Flag epitope was
inserted into the putative extracellular domain of the
GIRK4-200:GIRK1-194 chimera in order to assess its cell surface
localization. However, the Flag sequence was not efficiently recognized
by the anti-Flag antibody in the context of the GIRK4 extracellular
domain, and the anti-Flag antibody only weakly recognized the
GIRK4-200/GIRK1-194 chimera in permeabilized cells. The
GIRK4-200/GIRK1-194 chimera also contained the C-terminal AU5
epitope, which was used to assess the chimera's localization, since it
was efficiently recognized by the anti-AU5 antibody. GIRK4-200/GIRK1-194-expressing cells displayed a staining pattern consistent with the distribution of the endoplasmic reticulum of COS-7
cells (Fig. 7A). No obvious plasma membrane staining was
observed in permeabilized AU5-stained cells (compare Fig. 1D
and Fig. 7A, inset). The reverse chimera,
GIRK1-194:GIRK4-200, was expressed at levels comparable with that of
wild type GIRK1-Flag and GIRK4-AU1 and at the predicted molecular
weight (Fig. 7C, lane 5).
Immunolocalization of the GIRK1-194:GIRK4-200 chimera in
nonpermeabilized cells identified an average of 306 ± 39 (n = 2) positively staining cells, whereas 4460 ± 664 (n = 2) positive cells were identified after
permeabilization. The level of GIRK1-194:GIRK4-200 chimera staining
in nonpermeabilized cells was much lower than that of
GIRK1(N119D)-Flag/GIRK4-AU1-expressing cells. In permeabilized cells,
the GIRK1-194:GIRK4-200 chimera was plasma membrane-localized and
present in the endoplasmic reticulum (Fig. 7B; see
inset for higher magnification view). A summary of the
localization of the wild type and chimeric subunits studied here is
shown in Table I.
In an effort to identify the region within the GIRK4 C terminus that is
responsible for cell surface targeting of GIRK1/GIRK4 complexes, we
generated several GIRK4 deletion mutants, (Fig. 8, top). The deletion mutants
expressed the predicted size protein as assessed by Western blot (data
not shown). The ability of each GIRK4 deletion mutant to promote cell
surface localization of GIRK1 was assessed by coexpression with the
GIRK1(N119D)-Flag subunit and immunostaining in nonpermeabilized COS-7
cells. The GIRK4( GIRK4 Is Required for Cell Surface Localization of GIRK1 in
Vivo--
Wickman et al. (8) recently demonstrated that the
homozygous knockout of the GIRK4 gene in mice results in
decreased vagal and adenosine-mediated slowing of heart rate compared
with wild type littermates. GIRK4 knockout mice did not display any
detectable IKACh currents, supporting the finding that
GIRK1 alone does not substitute for wild type IKACh.
Although in atrial tissue, the mRNA levels of GIRK1 were unchanged,
in GIRK4 knockout mice, GIRK1 protein levels were decreased by
4-8-fold (8). An even more dramatic decrease in brain GIRK1 protein
was observed when the gene encoding GIRK2 was knocked out (40). To
determine the localization pattern of GIRK1 subunits in GIRK4 knockout
mice, enzymatically dissociated atrial myocytes from wild type and
GIRK4-knockout mice were immunostained with a GIRK1-specific antibody.
Antibodies that recognize the extracellular domain of native GIRK1 are
not available, so we utilized an antibody generated against the unique C-terminal domain of GIRK1 (anti-GIRK1; Ref. 11) in permeabilized cells. In wild type atrial cells, anti-GIRK1 immunostaining outlined the cell membrane with a small amount of perinuclear staining (Fig.
9, top right
panel). GIRK1 membrane staining was significantly reduced in
GIRK4 knockout myocytes with a concomitant increase in perinuclear
staining, indicating higher levels of intracellular GIRK1 (Fig. 9,
bottom right panel). Furthermore, we
consistently observed that the GIRK4 knockout atrial cells were more
compact and displayed more dense intracellular perinuclear structures under transmitted light illumination. These intracellular structures colocalized with GIRK1 immunostaining (Fig. 9, bottom
panels). The faint GIRK1 staining near the edge of GIRK4
knockout atrial cells (Fig. 9, arrow in bottom
right panel) may represent the endoplasmic
reticulum network that can extend throughout the cell and reach the
plasma membrane, or it may represent a small fraction of GIRK1 that is
still able to reach the cell surface in GIRK4 knockout atrial cells. We
assessed the glycosylation state of GIRK1 in GIRK4 knockout atrial
tissue by Western blot using three different GIRK1-specific polyclonal
antibodies. We did not detect any maturely glycosylated GIRK1 in GIRK4
knockout atrial membranes, (Fig. 9, bottom). In the original
characterization of GIRK1 levels in GIRK4 knockout mouse atrial tissue,
a small amount of fully glycosylated GIRK1 was noted (8). In the
current study, we found that the small amount of apparently
glycosylated GIRK1 previously seen was not GIRK1 but rather a protein
that migrates behind the upper third region of the diffuse glycosylated
GIRK1 band on SDS-PAGE and was nonspecifically recognized by the
anti-CSh antibody preparation (data not shown). Importantly, this
protein was not recognized by two separate GIRK1 antibodies directed
against either the N or C terminus of GIRK1. Therefore, our current
data, using several anti-GIRK1 antibodies, clearly shows that GIRK1 is
not glycosylated in GIRK4 knockout mice. The observed loss of maturely
glycosylated GIRK1 and absence of cell surface GIRK1 in GIRK4 knockout
myocytes indicates that GIRK4 plays a similar role in the maturation
and cell surface localization of IKACh in vivo
as is seen in transfected COS-7 cells.
We have demonstrated that GIRK4 subunits contain the information
necessary for appropriate processing and cell surface localization of
IKACh channels. We utilized antibodies directed against the Flag epitope, which was inserted into the extracellular domain of GIRK1
to determine if any GIRK1 homomultimeric channels were present on the
cell surface. GIRK1-expressing nonpermeabilized COS-7 cells displayed
no detectable GIRK1 at the cell surface despite high levels of protein
expression. Coexpression of GIRK4 and GIRK1 reproducibly resulted in
the appearance of robust cell surface GIRK1 staining. We also found
that fully glycosylated GIRK1 was observed only with coexpression of
GIRK1 with GIRK4. Since terminal glycosylation events mediated by
enzymes such as mannosidase II that occur in the Golgi apparatus (36,
41) probably produce fully glycosylated GIRK1, GIRK1 homotetramers are
likely to be present in a pre-Golgi compartment. The GIRK4 subunit
presumably allows movement of the heterotetramer through the Golgi on
its way to the cell surface. Alternatively, GIRK1 homotetramers enter
the Golgi but are not recognized by the Golgi-specific glycosyl-modifying enzymes and thus remain in a core-glycosylated state. The localization and processing of GIRK1 homotetramers is
reminiscent of the phenotype of the most common mutation found in the
cystic fibrosis transmembrane conductance regulator (CFTR) gene,
CFTR GIRK4 exerts its effects on heterotetramer processing and localization
at an early stage, since pulse-labeling experiments showed that GIRK1
and GIRK4 associate as early as 5 min following an
[35S]methionine pulse. This rapid association is
analogous to the association of Kv 1.1 and Kv 1.4 subunits (43) but is
distinct from the slow (hours) subunit association observed for
Na+ channel In our attempts to identify structural features of the GIRK1 subunit
responsible for its intracellular localization, we truncated the GIRK1
C terminus at amino acid 373 in order to delete the final 127 amino
acids. Although this deletion did not result in cell surface
localization of GIRK1, it did abolish the cytoskeletal staining pattern
observed for wild type GIRK1. The staining pattern of the
GIRK1( Several structure-function studies have implicated the unique
C-terminal domain of GIRK1 in G The GIRK1:GIRK4-pore chimera localized with a similar pattern to wild
type GIRK4, whereas the reverse chimera remained intracellular and was
expressed at low levels. The low expression levels of the
GIRK4:GIRK1-pore chimera probably resulted from misfolding, which
prevented us from drawing any conclusions from its localization pattern. Interpretation of the function and biochemical properties of
point-mutated and chimeric proteins can be confounded by significant changes in the three-dimensional structure of the altered protein. Misfolding of a mutant protein is often manifest by significant decreases in expression level as we observed for the GIRK4:GIRK1-pore chimera. Thus, some mutagenesis studies where only functional readouts
are measured should be interpreted with caution.
Increasing the contribution of the GIRK4 subunit in the
GIRK4:GIRK1-pore chimera to amino acid 199 (just past the predicted end
of the second transmembrane domain) resulted in the
GIRK4-200:GIRK1-194 chimera, which displayed expression levels
comparable with that of wild type GIRKs. Despite normal expression
levels, the GIRK4-200:GIRK1-194 chimera was not detectable on the
cell surface. Interestingly, the reverse GIRK1-194:GIRK4-200 chimera
showed obvious cell surface staining and was expressed at levels
comparable with that of wild type GIRKs. Analyzing the localization of
chimeras generated between GIRK1 and GIRK4 indicated that amino acids
194-501 of the GIRK1 C terminus caused intracellular localization of
GIRK1. Since the GIRK1( The correct folding and assembly of ion channel subunits into
functional oligomeric complexes involves the association of specific
intersubunit interactions sites (51). The absence of correct assembly
of intersubunit interaction sites would be predicted to cause
misfolding and therefore retention within the endoplasmic reticulum. We
would predict that the GIRK1 C terminus does not contain the structural
elements required for the proper formation of transportable
homotetrameric channels. The GIRK4 C terminus on the other
hand may contain the appropriate structural elements necessary for
formation of homotetramers and heterotetramers, which are compatible with cell surface targeting. Consistent with this
notion is evidence that the proximal C-terminal region of GIRK subunits
are involved in channel assembly (52). Our finding that the C terminus
of GIRK1 promotes intracellular localization is consistent with the
previous findings for GIRK1 and GIRK2 expressed in Xenopus
oocytes (53). A chimera between GIRK1 and GIRK2 subunits containing the
N and C termini of GIRK1 and membrane domains of GIRK2 showed less cell
surface staining compared with coexpressed wild type GIRK1/GIRK2
channels (53). Unlike the GIRK1-194:GIRK4-200 chimera, which
displayed significant cell surface staining in COS-7 cells, a chimera
containing the N and C termini of GIRK2 and the membrane domains of
GIRK1 did not show robust cell surface staining in Xenopus
oocytes (53). The presence of the GIRK1 N terminus, which may allow
appropriate folding of the GIRK1-194:GIRK4-200 chimera may contribute
to the enhanced cell surface staining seen in our study. Since COS-7
cells or CHO cells do not contain endogenous GIRK homologues (such as
the GIRK5/XIR subunit in Xenopus oocytes), they provide a
better system for studying homomultimeric GIRK channels. An alternative
explanation is that the C terminus of GIRK4 may contain a positive
signal for cell surface targeting. This signal may be encoded by a
unique linear amino acid sequence like the KDEL retention signal for
endoplasmic reticulum-localized proteins (54), or the signal may be
encoded by the three-dimensional structure achieved by the C-terminal
domain. The absence of a cell surface localization signal in the GIRK1
C terminus would result in intracellular retention by default. Finally,
it is possible that an as yet unknown protein is involved in GIRK trafficking.
Based on our data, the simplest explanation for the absence of
functional IKACh-like channel activity in
GIRK1-only-expressing mammalian cell lines is that the subunits are
mislocalized. A previous report suggested that GIRK1 homomultimers are
nonfunctional because of the presence of a unique phenylalanine residue
at position 137 (55). A F137S mutant GIRK1 subunit expressed
G The marked decrease in total GIRK1 protein and intracellular
redistribution of GIRK1 in GIRK4 knockout atrial myocytes and the loss
of any detectable maturely glycosylated GIRK1 indicate that GIRK4
subunits function to ensure appropriate processing and cell surface
localization of IKACh in vivo. It is difficult to rule out the possibility that a small fraction of GIRK1 subunits may
exist at the cell surface in GIRK4 knockout myocytes, since an antibody
directed against the extracellular domain of native GIRK1 is not
available. However, since inside-out patches from GIRK4-knockout
myocytes do not show any detectable IKACh-like channel
activity (8) and there is no detectable mature form of GIRK1 present in
GIRK4 knockout atrial tissue, it is unlikely that any GIRK1 is cell
surface-localized in GIRK4 knockout mice.
It would appear that GIRK4 plays a major role in both processing and
G-protein activation of IKACh. That leaves open the
question of the role of GIRK1 subunits. GIRK1 may play a role in
determining IKACh single channel kinetics, since
GIRK1/GIRK4 channels have much longer open times than GIRK4 alone (11)
and may provide sites for channel modulation through protein kinases.
Future studies should allow elucidation of the mechanism by which the
discrete GIRK4 domain from amino acids 375-399 allow cell surface
localization of IKACh.
subunits. The GIRK1 subunit is atypical in the GIRK family in having a
unique (~125-amino acid) domain in its distal C terminus. GIRK1
cannot form functional channels by itself but must combine with another
GIRK family member (GIRK2, GIRK3, or GIRK4), which are themselves
capable of forming functional homotetramers. Here we show, using
an extracellularly Flag-tagged GIRK1 subunit, that GIRK1 requires
association with GIRK4 for cell surface localization. Furthermore,
GIRK1 homomultimers reside in core-glycosylated and nonglycosylated
states. Coexpression of GIRK4 caused the appearance of the mature
glycosylated form of GIRK1. [35S]Methionine
pulse-labeling experiments demonstrated that GIRK4 associates with
GIRK1 either during or shortly after subunit synthesis. Mutant and
chimeric channel subunits were utilized to identify domains responsible
for GIRK1 localization. Truncation of the unique C-terminal domain of
374-501 resulted in an intracellular GIRK1 subunit that produced
normal IKACh-like channels when coexpressed with GIRK4.
Chimeras containing the C-terminal domain of GIRK1 from amino acid 194 to 501 were intracellularly localized, whereas chimeras containing the
C terminus of GIRK4 localized to the cell surface. Deletion analysis of
the GIRK4 C terminus identified a 25-amino acid region required for
cell surface targeting of GIRK1/GIRK4 heterotetramers and a 25-amino
acid region required for cell surface localization of GIRK4
homotetramers. GIRK1 appeared intracellular in atrial myocytes isolated
from GIRK4 knockout mice and was not maturely glycosylated, supporting
an essential role for GIRK4 in the processing and cell surface
localization of IKACh in vivo.
INTRODUCTION
Top
Abstract
Introduction
References
- and
-subunits. It is
the G
subunits that activate IKACh and
hyperpolarize the cell, thereby slowing heart rate (4, 8).
activates recombinant GIRK1/GIRK4 channels, and
this activation is likely to be mediated by direct binding of G
subunits to GIRK1 and GIRK4 subunits (13-17). The
GIRK4 subunit plays a pivotal role in G
-mediated
activation of IKACh, since mutation of specific amino acids
located within residues 216-248 of GIRK4 either altered the potency of
G
or abolished entirely the G
activation of GIRK1/GIRK4 mutant channels (18). GIRK1/GIRK4 channels
are also modulated by phosphorylation (19), intracellular
Na+ ions (20), and mechanical stress. Furthermore, the
G
-mediated activation of GIRK1/GIRK4 may require the
presence of the rare membrane phospholipid,
phosphatidylinositol-4,5-bisphosphate (21, 22).
SYG) in the K+ selectivity sequence of the GIRK2-1
subunit results in a nonselective, constitutively active GIRK1/GIRK2
channel, which is responsible for the cerebellar granule cell death and
consequent severe ataxia of the weaver mouse (29-32).
-dependent K+ currents that
are identical to IKACh (11, 33, 34). Like native
IKACh, GIRK1/GIRK4 heterotetramers manifest single channels with 1-2-ms open times and a ~35-pS conductance (11). CHO or COS-7
cells expressing only GIRK1 subunits do not display any IKACh-like channel activity (11, 35) despite high levels of protein expression and formation of homotetramers (12). Although expression of GIRK1 alone in Xenopus oocytes results in a
low level of IKACh-like channel activity, this activity has
been attributed to the presence of an endogenous oocyte GIRK homologue
termed GIRK5 (XIR; Ref. 34). CHO cells, COS-7 cells, or
Xenopus oocytes expressing GIRK4 homotetramers do give rise
to G
-activated inwardly rectifying K+
channels with atypical, small amplitude single channels that have yet
to be observed in native cells (11, 33). Furthermore, isolated cardiac
atrial cells from GIRK4 knockout mice do not display any detectable
IKACh channel activity (8). These observations suggest a
critical role for GIRK4 in the expression of IKACh channels in heterologous cells and in vivo.
EXPERIMENTAL PROCEDURES
80 mV. The
currents were amplified using an Axopatch 200A amplifier (Axon
Instruments, Foster City, CA) and stored on a VCR tape after filtering
at 5 kHz (four-pole low pass Bessel filter). The signal was then
digitally sampled at 50 kHz frequency and stored on a computer hard
disk using pClamp6 software (Axon Instruments, Foster City, CA). While no further filtering has been performed for kinetic and
nPo analysis, data displayed in Fig. 5 have been
low pass-filtered at 2 kHz frequency with an eight-pole Bessel filter.
For each patch, nPo has been determined from
10-s data segments before and after the addition of G
to the bath (final G
concentration 20 nM). pClamp6 software was used for data analysis. Mean open time was determined from idealized tracings using custom-made software
written in TurboPascal. We chose this method for comparing the wild
type and truncated channel rather than exponential fitting because most
of the patches contained multiple simultaneous channel openings.
374-501)-AU5 with
the GIRK4-AU1 subunit, cells were incubated overnight with 50 µCi/ml
[35S]methionine followed by immunoprecipitation as
described above. Samples were analyzed on SDS-PAGE followed by
fluorography with Amplify (Amersham Pharmacia Biotech) and autoradiography.
RESULTS
-activated single channels with short (atypical)
duration and variable amplitude (11, 33). Our previous studies
identified strikingly different localization patterns for individually
expressed epitope-tagged GIRK1-AU5 and GIRK4-AU1 subunits in COS-7
cells (35). We postulated that the differences in functional expression
of GIRK1 versus GIRK4 subunits could be due to the inability
of GIRK1 to be trafficked to the cell surface. To test this hypothesis,
we inserted the Flag epitope (DYKDDDDK) into the putative extracellular
domain of GIRK1(GIRK1-Flag) between amino acids 114 and 115. This
GIRK1-Flag subunit should be detectable by the anti-Flag antibody in
nonpermeabilized cells only when it is present at the cell surface. The
N-linked glycosylation consensus sequence at position 119 was eliminated by an N119D mutation to prevent potential obstruction of
the epitope by nearby sugar moieties. The GIRK1(N119D)-Flag subunit
gave currents similarly to wild type GIRK1 when coexpressed with GIRK4
in COS-7 cells (data not shown). Nonpermeabilized COS-7 cells
expressing the GIRK1(N119D)-Flag subunit did not display any specific
immunofluorescence staining (Fig.
1A) compared with pCDNA3
transfected controls, (GIRK1(N119D)-Flag, 142 ± 71 cells;
n = 5 coverslips; versus pCDNA3, 139 ± 27 cells; n = 4 coverslips). The staining
pattern in nonpermeabilized pCDNA3-transfected and
GIRK1(N119D)-Flag-transfected cells was quantitatively lower and
irregular compared with the anti-Flag staining seen in
GIRK1(N119D)-Flag/GIRK4-AU1-expressing nonpermeabilized cells. We
conclude that the staining was due to nonspecific binding of the M2
antibody. Permeabilization of the GIRK1(N119D)-Flag-transfected COS-7
cells revealed a large number of GIRK1-expressing cells (2623 ± 426 S.D. cells; n = 3) with a cytoskeletal staining
pattern (Fig. 1B). Coexpression of GIRK4-AU1 with
GIRK1(N119D)-Flag resulted in readily identifiable
GIRK1(N119D)-Flag-positive cells (1557 ± 452 cells;
n = 3) under nonpermeabilized conditions (Fig.
1C), indicating that GIRK1 requires coexpression of GIRK4
for cell surface localization. The staining pattern of cell surface
GIRK1 was fairly evenly distributed across the cell membrane,
suggesting that the cell surface GIRK1-Flag/GIRK4-AU1 complexes were
not associated with the cytoskeleton. A significant amount of
intracellularly localized GIRK1 was still seen in permeabilized
anti-Flag-stained GIRK1(N119D)-Flag/GIRK4-AU1-transfected cells (Fig.
1D).
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Fig. 1.
GIRK4 allows cell surface expression of
GIRK1. A, nonpermeabilized COS-7 cells expressing
GIRK1(N119D)-Flag. B, permeabilized COS-7 cells expressing
the GIRK1(N119D)-Flag subunit. C, nonpermeabilized COS-7
cells coexpressing GIRK1(N119D)-Flag and GIRK4-AU1 subunits.
D, permeabilized COS-7 cells coexpressing GIRK1(N119D)-Flag
and GIRK4-AU1 subunits. All immunostaining was performed using a 1:100
dilution of the M2 anti-Flag monoclonal antibody and detected using a
1:200 dilution of donkey anti-mouse rhodamine-conjugated secondary
antibody. The scale bar in C also
applies to A, B, and D. Images were
obtained from a Zeiss LSM 410 confocal microscope using a 568-nm
excitation wavelength with a long pass 590-nm emission filter. The
pinhole size used was 16, and the contrast/brightness settings were
similar for each image. The confocal section was 1.5 µm thick. In
cases were there was no staining, the field was z-sectioned
across 20 µm.
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Fig. 2.
Glycosylation state of GIRK1 is determined by
GIRK4. A, lanes 1-3, Western blot of equal
amounts of total membrane protein from GIRK1-Flag/GIRK4-AU1-expressing
COS cells treated with buffer (control), endoglycosidase F, or
endoglycosidase H using a GIRK1-specific polyclonal antibody.
Lanes 4 and 5, Western blot of total
membrane protein from wild type or N119D mutant GIRK1-Flag-expressing
COS-7 cells. Lower graph, densitometric scans of
equal areas from control, endoglycosidase H-treated, or endoglycosidase
F-treated lanes. B, immunoprecipitations from GIRK1-AU5,
GIRK4-AU1, or GIRK1-AU5/GIRK4-AU1-expressing COS-7 cells immunoblotted
with GIRK1 and GIRK4-specific anti-N-terminal peptide antibodies.
C, COS-7 cells cotransfected with GIRK1-AU5 and GIRK4-AU1
and pulse-labeled with [35S]methionine for the indicated
times. Coimmunoprecipitation was performed with the AU1 antibody
followed by analysis on a 10% SDS-PAGE, fluorography, and
autoradiography.
Activation--
GIRK1's inability to be trafficked to the cell
surface was unexpected, since GIRK1 shares significant homology with
cell surface-localized GIRK4 (45% identity and 58% similarity). We
mutated the GIRK1 cDNA in order to identify regions within the
subunit that might be responsible for its intracellular localization in
the absence of coexpressed GIRK4 subunits. Amino acids 373-501 of
GIRK1 represent the terminal 127 amino aicds of GIRK1 and are unique
within the entire IRK family of proteins. Insertion of the AU5 epitope
and stop codon at amino acid 373 produced a truncated form of GIRK1, GIRK1(
374-501)-AU5, with a predicted molecular mass of
approximately 42 kDa (Fig. 3,
lane 1) in [35S]methionine-labeled
cells. The GIRK1(
374-501)-AU5 protein migrated as a doublet
suggesting it was core-glycosylated, like the full-length GIRK1
subunit. GIRK1(
374-501)-AU5 subunits were coimmunoprecipitated by
the GIRK4-AU1 subunit, since the AU1 antibody does not directly interact with the GIRK1(
374-501)-AU5 protein, (Fig. 3,
lane 2). Immunolocalization of the
GIRK1(
374-501)-AU5 subunit revealed a loss of the cytoskeletal
staining pattern (Fig. 4A), as
seen for the wild type GIRK1-AU5 subunit (Fig. 4A,
inset). The staining pattern for the GIRK1(
374-501)-AU5
subunit is consistent with the extensive tubular network of the COS
cell endoplasmic reticulum (38, 39). Further truncations of the GIRK1 C
terminus up to amino acid 215 were generated in the GIRK1-Flag cDNA
and immunostained in nonpermeabilized COS-7 cells. None of the
truncated GIRK1-Flag subunits were localized at the cell surface in
nonpermeabilized cells (data not shown). The observed shift in
localization for the GIRK1(
374-501)-AU5 subunit suggested that this
region of GIRK1 may be involved in the cytoskeletal localization of
GIRK1. To test whether the region of amino acids 373-501 of GIRK1
played a dominant role in the localization of GIRK1 to the
cytoskeleton, we assessed the staining pattern of 1) amino acids
373-501 expressed independently of the rest of the GIRK1 protein
(Met-373-501-Flag) and 2) a chimeric channel subunit with amino acids
373-501 of GIRK1 fused to the C terminus of the non-cytoskeletally
localized GIRK4 subunit (Fig. 4B), GIRK4:GIRK1-(373-501).
Representative examples of cytoskeletal localization of GIRK1-AU5 (Fig.
4A, inset) and the localization of GIRK4-AU1
(Fig. 4B) are shown for comparison. The Met-373-501-Flag
protein displayed high expression levels as assessed by immunostaining.
Shown in Fig. 4C is a confocal image of the staining pattern
of an anti-Flag-stained Met-373-501-Flag-expressing COS-7 cell.
Significant staining is seen in the cytoplasm and nucleus. A lower
amount of cytoskeletal staining was consistently observed in
Met-373-501-Flag-expressing cells, compared with GIRK1-AU5-expressing cells (Fig. 4A, inset). The
GIRK4:GIRK1-(373-501) chimeric subunit displayed an immunostaining
pattern similar to that of wild type GIRK4-AU1 (Fig. 4, compare
D and B). In some cells, the chimera demonstrated
weak cytoskeletal staining. These data suggest that the presence of
amino acids 373-501 of GIRK1 are not sufficient to promote the
intracellular retention of GIRK4, since the GIRK4:GIRK1-(373-501) chimera localized similarly to the GIRK4-AU1 subunit. These residues do
not play a dominant role in cytoskeletal localization. The lower degree
of cytoskeletal localization of the Met-373-501-Flag and
GIRK4:GIRK1-(373-501) constructs suggests that residues 373-501 of
GIRK1 best function to promote cytoskeletal localization in the context
of the intact GIRK1 protein.
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Fig. 3.
Subunit association of
GIRK1( 374-501)-AU5 and GIRK4-AU1.
Transfection of GIRK1(
374-501)-AU5 or
GIRK4-AU1/GIRK1(
374-501)-AU5 followed by
[35S]methionine labeling. Lane 1,
immunoprecipitation of GIRK1(
374-501)-AU5 with the AU5 monoclonal
antibody; lane 2, coimmunoprecipitation of
GIRK4-AU1/GIRK1(
374-501)-AU5 using the AU1 monoclonal
antibody.
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Fig. 4.
Assessment of residues 373-501 of GIRK1 in
subunit localization. A, a representative example of
AU5 immunostaining of permeabilized COS-7 cells expressing the
GIRK1( 374-501)-AU5 subunit. Inset, AU5 immunostaining of
permeabilized COS-7 cells expressing the wild type GIRK1-AU5 subunit.
B, wild type GIRK4-AU1-expressing COS-7 cells stained with
the anti-AU1 antibody. C, anti-Flag-stained, permeabilized
COS-7 cells expressing the Met-373-501-Flag GIRK1 C-terminal domain.
D, AU5-stained COS-7 cells expressing the
GIRK4:GIRK1-(373-501) chimera. A 1:200 dilution of donkey anti-mouse
rhodamine-conjugated secondary antibody was used for detection. The
scale for B and D is the same as for A
and C, respectively. The schematic diagram at the
bottom represents constructs used in experiments. Images
were obtained using a Zeiss LSM 410 confocal microscope using a 568-nm
excitation wavelength with a long pass 590-nm emission filter. The
pinhole size used was 16, and the contrast/brightness settings were
similar for each image. The confocal section was 1.5 µm thick.
374-501)-AU5 subunit in the presence of 20 nM G
(data not shown). Coexpression of the GIRK1(
374-501)-AU5 and wild type GIRK4-AU1 gave rise to robust G
-activated single channels in inside-out patches
from COS-7 cells (Fig. 5). The single
channel conductance (~35 pS) and mean open time (~1.5 ms) were
similar to that of native atrial IKACh. Channel activity
before the G
addition was 3.8 times lower for
GIRK1-AU5/GIRK4-AU1 (0.0074 ± 0.052 pA; n = 6 patches) compared with GIRK1(
374-501)-AU5/GIRK4-AU1-expressing cells (0.0282 ± 0.202 pA; n = 7). However, this
difference was not significantly different when compared in an unpaired
t test.
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Fig. 5.
GIRK1( 374-501)-AU5/GIRK4-AU1
channels are activated by G-protein
G
subunits and
display IKAch single channel properties. Shown is an
inside-out patch from a GIRK1(
374-501)-AU5/GIRK4-AU1
cotransfected COS-7 cell. The bar denotes the addition
of 20 nM G
. The holding potential was
80 mV, and recordings were performed in symmetrical 140 mM [K+].
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Fig. 6.
Localization of GIRK1, GIRK4-pore domain
chimeras. A, GIRK4-AU1 staining in permeabilized
(+TX-100) COS-7 cells using a 1:100 dilution of anti-AU1
antibody. B, GIRK1-Flag staining in permeabilized COS-7
cells using a 1:100 dilution of anti-Flag M2 antibody. C,
nonpermeabilized GIRK1:GIRK4-pore chimera expressing COS-7 cells.
D, permeabilized GIRK1:GIRK4-pore expressing COS-7 cells
stained with anti-Flag M2 antibody. E, nonpermeabilized
COS-7 cells expressing the GIRK4:GIRK1-pore chimera stained with
anti-Flag M2 antibody. F, permeabilized COS-7 cells
expressing the GIRK4:GIRK1-pore chimera. A 1:200 dilution of donkey
anti-mouse rhodamine-conjugated secondary antibody was used for
detection. Images were obtained using a Zeiss LSM 410 confocal
microscope using a 568-nm excitation wavelength with a long pass 590-nm
emission filter. The pinhole size used was 16, and the
contrast/brightness settings were similar for each image. The confocal
section was 1.5 µm thick.
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Fig. 7.
Localization of GIRK1, GIRK4 C-terminal
chimeras. A, localization of the GIRK4-200:GIRK1-194
chimera in permeabilized COS-7 cells using the anti-AU5 antibody.
Inset, ~2.5-fold magnification of boxed
area. B, immunolocalization of
GIRK1-194:GIRK4-200 chimera in permeabilized COS-7 cells using the
anti-Flag M2 antibody. Inset, ~3-fold magnification of
boxed area. Images were obtained using a Zeiss
LSM 410 confocal microscope using a 568-nm excitation laser line with a
long pass 590-nm emission filter. The pinhole size used was 16, and the
contrast/brightness settings were similar for each image. The confocal
section was 1.5 µm thick. C, equal amounts of membranes
from COS-7 cells transfected with the indicated wild type or chimeric
subunits were analyzed by SDS-PAGE and Western blotting with either a
GIRK1-specific antipeptide antibody raised against an N-terminal
peptide from GIRK1 (lanes 1, 3, and
5) or an antibody that recognized the N terminus of GIRK4
(lanes 2, 4, 6, and
7).
Cell surface localization of wild type and mutant/chimeric subunits
400-419) deletion mutant was able to localize to
the cell surface as a homotetramer and promote cell surface
localization of GIRK4(
400-419)/GIRK1(N119D)-Flag heterotetramers,
(Fig. 8, bottom panel). The GIRK4(
375-419)
deletion mutant also localized to the cell surface as a homotetramer
displaying a staining pattern identical to that of wild type GIRK4
(Fig. 8, bottom panel); however, the
GIRK4(
375-419) subunit did not promote cell surface localization of
GIRK1(N119D)-Flag (Fig. 8, bottom panel).
Interestingly, in permeabilized cells coexpressing GIRK4(
375-419)
and GIRK1(N119D)-Flag and stained with the AU1 antibody,
GIRK4(
375-419) was observed both at the plasma membrane and
cytoskeleton (Fig. 8, bottom panel). The
cytoskeletal staining is indicative of GIRK4(
375-419) interaction with the GIRK1(N119D)-Flag protein. Furthermore, homotetrameric GIRK4(
400-419) and GIRK4(
375-419) subunits were activated by 20 nM
G
.2 The
ability of the GIRK4(
375-419) subunit to interact with the GIRK1(N119D)-Flag subunit and be activated by G
indicates that we have not perturbed subunit association or G-protein
activation but rather selectively blocked the targeting of the
heterotetramer to the cell surface. Further deletion of amino acids
350-419 of GIRK4 (GIRK4(
350-419)) resulted in a loss of both homo-
and heterotetramer targeting to the cell surface. An example of the
intracellular localization of the GIRK4
300-419 mutant is shown in
Fig. 8 (bottom panel). Taken together, these data
suggest that amino acids 375-399 are critical for heterotetramer cell
surface targeting while amino acids 350-374 are critical for
homotetramer formation.
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Fig. 8.
Amino acids 375-399 of GIRK4 are required
for cell surface localization of GIRK1/GIRK4 heterotetramers, whereas
amino acids 350-374 are required for both homotetramer and
heterotetramer cell surface localization. Top
panel, schematic diagram showing the
deletions of GIRK4 analyzed and a summary of their localization
patterns. Each construct contained the AU1 epitope (DTYRYI) at the C
terminus. Images of the localization pattern for the constructs shown
in the bottom panel are marked with a
filled square. Bottom
panel, immunolocalization of GIRK1(N119D)-Flag in
nonpermeabilized COS-7 coexpressing the indicated GIRK4 deletion
construct and GIRK1(N119D)-Flag. Lower four
images, permeabilized COS-7 cells expressing the indicated
GIRK4 subunits alone and immunostained with the anti-AU1 antibody. The
light gray shading depicts the GIRK4
region essential for heterotetramer localization, and the
dark gray shading represents the
region essential for GIRK4 homotetramer localization. The pinhole size
used was 16, and the contrast/brightness settings were similar for each
image The confocal section was 1.5 µm thick.
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Fig. 9.
Immunolocalization of GIRK1 subunits in wild
type and GIRK4 knockout mouse dissociated atrial myocytes.
Top panels, enzymatically dissociated wild type
mouse atrial cells immunostained with a 2 µg/ml concentration of a
GIRK1-specific polyclonal antibody raised against the unique C terminus
of GIRK1. Left panel, transmitted light image of
the field imaged for GIRK1 fluorescence. Right
panel, confocal image of GIRK1-stained atrial cells.
Bottom panels, enzymatically dissociated GIRK4
knockout mouse atrial cells immunostained using 2 µg/ml of a
GIRK1-specific polyclonal antibody. Left panel,
transmitted light image of the field imaged for fluorescence.
Right panel, confocal image of GIRK1-stained
atrial cells. The arrowheads indicate the cell's edge.
Primary antibodies were detected using a 1:200 dilution of
rhodamine-conjugated goat anti-rabbit secondary antibody. Images were
obtained using a Zeiss LSM 410 confocal microscope using a 568-nm
excitation wavelength with a long pass 590-nm emission filter. The
pinhole size used was 16, and the contrast/brightness settings were
similar for each image. The confocal section was 1.5 µm thick.
DISCUSSION
F508. The CFTR
F508 protein is a
temperature-sensitive mutant that is incompletely glycosylated and
remains in the endoplasmic reticulum (42). In addition, a large
fraction, ~75%, of wild type CFTR is intracellular (42). Unlike
CFTR
F508, the intracellular localization of GIRK1 subunits are not
temperature-sensitive, since no cell surface GIRK1(N119D)-Flag was
detected when expressing cells were incubated at 25 °C for several
days (data not shown).
,
1, and
2
subunits (44). Studies examining the assembly and processing of
voltage-gated K+ channels revealed a role for
-subunits
in the processing and efficient cell surface localization of Kv 1.2 subunits (45). Whereas association of the Kv 1.2
-subunit with its
cytosolic
-subunit increased the degree of fully glycosylated and
cell surface-localized Kv 1.2
-subunits, in the present study we
observed a complete absence of detectable mature glycosylation or cell surface localization of GIRK1 without coexpressed GIRK4. Like GIRK1 and
GIRK4 subunits, Drosophila Shaker K+ channel
- and
-subunits were shown to assemble in the endoplasmic reticulum (46). In contrast to the
-subunits of mammalian
voltage-gated K+ channels (45), the Shaker K+
channel's maturation and localization were not altered by coexpressed
-subunits (46). We conclude that one of the pore-forming subunits, GIRK4, appears to serve a role in the processing of IKACh
analogous to that of the Kv
-subunits associated with Kv 1.2.
374-501)-AU5 subunit was consistent with retention in the
endoplasmic reticulum. To test if amino acids 374-501 of GIRK1 could
independently localize to the cytoskeleton, we created a cDNA
representing Met-373-501-Flag of the GIRK1 subunit. The Met-373-501-Flag protein localized throughout the cell with a small
degree of cytoskeletal staining. Furthermore, a chimeric channel
containing the full sequence of GIRK4 with GIRK1 amino acids
374-501-AU5 added to the C terminus localized primarily to the cell
surface of COS-7 cells and showed a small degree of cytoskeletal
staining. More extensive chimeras generated between GIRK1 and GIRK4 did
not display cytoskeletal staining like that seen for wild type GIRK1.
Taken together, these data suggest that although amino acids 374-501
are necessary for cytoskeletal localization of GIRK1, they do not act
as an autonomous signal for this localization pattern. The relationship
of the GIRK1 cytoskeletal staining to IKACh function
remains to be determined. The lack of any GIRK1 cytoskeletal staining
in isolated atrial myocytes suggests that the cytoskeletal staining
might be a unique feature of heterologously expressed GIRK1 or it may
represent a physiologic interaction that is exaggerated by
overexpression in COS-7 cells.
activation of
IKACh. In this study, we found normal G
activation of heteromultimeric channels lacking this domain. The lack
of involvement of this unique region of GIRK1 in G-protein activation
in this study is contradictory to findings published previously, which
proposed that this region of GIRK1 is a critical determinant for
G
-mediated activation of recombinant (14, 16, 17, 47,
48) and native IKACh (49). A previous study identified a
peptide composed of amino acids 482-498 of GIRK1 that blocked
GIRK1/GIRK4 channel activity in Xenopus oocytes (50). The
authors concluded that the peptide represented part of the gate that
kept GIRK1/GIRK4 channels inactive until bound by G
subunits. One would predict that a GIRK1 subunit with this domain
deleted would have a high basal activity; however, GIRK1 mutant
subunits with the identified domain deleted did not express functional
channels (50). The GIRK1(
374-501)-AU5 represents a logical
construct to use to test this hypothesis, since it lacks amino acids
482-498 and expresses functional channels. Although the
GIRK1(
374-501)-AU5/GIRK4-AU1 channel displayed somewhat higher
basal activity compared with wild type GIRK1-AU5/GIRK4-AU1 channels,
this difference was not statistically significant.
374-501)-AU5 subunit did not localize to the
cell surface, we can narrow the critical region for intracellular
localization of GIRK1 homotetramers to residues 194-373 of GIRK1.
Furthermore, the presence of amino acids 200-419 of GIRK4 in chimeric
subunits resulted in cell surface localization. Deletion analysis of
GIRK4 identified a C-terminal region of GIRK4 (amino acids 375-399) that is required for cell surface localization of GIRK1/GIRK4 heterotetramers but did not affect cell surface localization of GIRK4(
375-419) homotetramers. Deletion of amino acids 350-419 prevented both GIRK4 homotetramers and GIRK1/GIRK4(
350-419)
channels from being delivered to the cell surface.
-activated channels in inside-out patches from
cRNA-injected Xenopus oocytes (55). GIRK1(F137S) mutant
channels displayed a unique single channel conductance of 15.4 pS (55).
However, the F137S-mutated GIRK1-Flag subunit did not produce
detectable cell surface GIRK1 in nonpermeabilized in COS-7 cells (data
not shown). In Xenopus oocytes, cell surface localization
was observed for both a GIRK1-green fluorescent protein fusion protein
and F137S mutant GIRK1-green fluorescent protein fusion protein (56).
An earlier report, published before the identification of GIRK5/XIR,
reported that in Xenopus oocytes the GIRK1(F137S) mutant
displayed much faster activation kinetics in macroscopic current
recordings (7). The functional homomultimers observed for the
GIRK1(F137S) mutant in Xenopus oocytes may represent
differences in subunit processing between oocytes versus
mammalian cell expression systems or the influence of XIR. Without
electrophysiological measurements in cells expressing cell surface
GIRK1 homotetramers, we cannot rule out that GIRK1 homotetramers may
lack the determinants necessary to function as GIRK channels. This
would be consistent with our previous mutagenesis study, which
identified key amino acid residues in GIRK4 that are required for
G
-activation and failed to identify any similar
residues in GIRK1 (18).
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ACKNOWLEDGEMENTS |
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We are grateful to Grigory Krapivinsky for providing GIRK1- and GIRK4-specific antibodies. The technical assistance of Matthew Burtelow is greatly appreciated. We thank Mike Badminton and Loren Runnels for helpful discussions and Loren Runnels for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health (NIH) Grant 34873 (to D. E. C.), the Howard Hughes Medical Institute (to D. E. C. and M. E. K.), the Mayo Foundation (to J. N. and S. C.), and NIH Training Grant T32HL07572-13 (to K. W.).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.
To whom correspondence should be addressed: Howard Hughes
Medical Institute, Enders 1309, 320 Longwood Ave., Children's
Hospital, Boston, MA 02115. Tel.: 617-355-6163; Fax: 617-730-0692;
E-mail: clapham{at}rascal.med.harvard.edu.
The abbreviations used are: IRK, inwardly rectifying K+ channel; IKACh, acetylcholine-activated potassium current; ACh, acetylcholine; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonic acid; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; CFTR, cystic fibrosis transmembrane conductance regulator.
2 M. E. Kennedy, J. Nemec, and D. E. Clapham, unpublished observation.
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REFERENCES |
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