GIRK4 Confers Appropriate Processing and Cell Surface Localization to G-protein-gated Potassium Channels*

Matthew E. KennedyDagger §, Jan Nemec, Shawn Corey, Kevin Wickman§, and David E. ClaphamDagger §parallel

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
References

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 Gbeta gamma 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 Delta 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

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 alpha - and beta gamma -subunits. It is the Gbeta gamma subunits that activate IKACh and hyperpolarize the cell, thereby slowing heart rate (4, 8).

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).

Gbeta gamma activates recombinant GIRK1/GIRK4 channels, and this activation is likely to be mediated by direct binding of Gbeta gamma subunits to GIRK1 and GIRK4 subunits (13-17). The GIRK4 subunit plays a pivotal role in Gbeta gamma -mediated activation of IKACh, since mutation of specific amino acids located within residues 216-248 of GIRK4 either altered the potency of Gbeta gamma or abolished entirely the Gbeta gamma 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 Gbeta gamma -mediated activation of GIRK1/GIRK4 may require the presence of the rare membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (21, 22).

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 right-arrow 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).

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 Gbeta gamma -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 Gbeta gamma -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.

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.

    EXPERIMENTAL PROCEDURES

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 -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 Gbeta gamma to the bath (final Gbeta gamma 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.

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(Delta 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

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 Gbeta gamma -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.

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.


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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.

The Unique GIRK1 C-terminal Domain Is Required for Cytoskeletal Localization of GIRK1 and Is Not Required for Gbeta gamma 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(Delta 374-501)-AU5, with a predicted molecular mass of approximately 42 kDa (Fig. 3, lane 1) in [35S]methionine-labeled cells. The GIRK1(Delta 374-501)-AU5 protein migrated as a doublet suggesting it was core-glycosylated, like the full-length GIRK1 subunit. GIRK1(Delta 374-501)-AU5 subunits were coimmunoprecipitated by the GIRK4-AU1 subunit, since the AU1 antibody does not directly interact with the GIRK1(Delta 374-501)-AU5 protein, (Fig. 3, lane 2). Immunolocalization of the GIRK1(Delta 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(Delta 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(Delta 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(Delta 374-501)-AU5 and GIRK4-AU1. Transfection of GIRK1(Delta 374-501)-AU5 or GIRK4-AU1/GIRK1(Delta 374-501)-AU5 followed by [35S]methionine labeling. Lane 1, immunoprecipitation of GIRK1(Delta 374-501)-AU5 with the AU5 monoclonal antibody; lane 2, coimmunoprecipitation of GIRK4-AU1/GIRK1(Delta 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(Delta 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.

No channel activity was observed in inside-out patches from COS-7 cells expressing the GIRK1(Delta 374-501)-AU5 subunit in the presence of 20 nM Gbeta gamma (data not shown). Coexpression of the GIRK1(Delta 374-501)-AU5 and wild type GIRK4-AU1 gave rise to robust Gbeta gamma -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 Gbeta gamma addition was 3.8 times lower for GIRK1-AU5/GIRK4-AU1 (0.0074 ± 0.052 pA; n = 6 patches) compared with GIRK1(Delta 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(Delta 374-501)-AU5/GIRK4-AU1 channels are activated by G-protein Gbeta gamma subunits and display IKAch single channel properties. Shown is an inside-out patch from a GIRK1(Delta 374-501)-AU5/GIRK4-AU1 cotransfected COS-7 cell. The bar denotes the addition of 20 nM Gbeta gamma . The holding potential was -80 mV, and recordings were performed in symmetrical 140 mM [K+].

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).


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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).

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.

                              
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Table I
Cell surface localization of wild type and mutant/chimeric subunits

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(Delta 400-419) deletion mutant was able to localize to the cell surface as a homotetramer and promote cell surface localization of GIRK4(Delta 400-419)/GIRK1(N119D)-Flag heterotetramers, (Fig. 8, bottom panel). The GIRK4(Delta 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(Delta 375-419) subunit did not promote cell surface localization of GIRK1(N119D)-Flag (Fig. 8, bottom panel). Interestingly, in permeabilized cells coexpressing GIRK4(Delta 375-419) and GIRK1(N119D)-Flag and stained with the AU1 antibody, GIRK4(Delta 375-419) was observed both at the plasma membrane and cytoskeleton (Fig. 8, bottom panel). The cytoskeletal staining is indicative of GIRK4(Delta 375-419) interaction with the GIRK1(N119D)-Flag protein. Furthermore, homotetrameric GIRK4(Delta 400-419) and GIRK4(Delta 375-419) subunits were activated by 20 nM Gbeta gamma .2 The ability of the GIRK4(Delta 375-419) subunit to interact with the GIRK1(N119D)-Flag subunit and be activated by Gbeta gamma 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(Delta 350-419)) resulted in a loss of both homo- and heterotetramer targeting to the cell surface. An example of the intracellular localization of the GIRK4Delta 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.

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.


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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

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, CFTRDelta F508. The CFTRDelta 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 CFTRDelta 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).

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 alpha , beta 1, and beta 2 subunits (44). Studies examining the assembly and processing of voltage-gated K+ channels revealed a role for beta -subunits in the processing and efficient cell surface localization of Kv 1.2 subunits (45). Whereas association of the Kv 1.2 alpha -subunit with its cytosolic beta -subunit increased the degree of fully glycosylated and cell surface-localized Kv 1.2 alpha -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 alpha - and beta -subunits were shown to assemble in the endoplasmic reticulum (46). In contrast to the beta -subunits of mammalian voltage-gated K+ channels (45), the Shaker K+ channel's maturation and localization were not altered by coexpressed beta -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 beta -subunits associated with Kv 1.2.

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(Delta 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.

Several structure-function studies have implicated the unique C-terminal domain of GIRK1 in Gbeta gamma activation of IKACh. In this study, we found normal Gbeta gamma 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 Gbeta gamma -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 Gbeta gamma 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(Delta 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(Delta 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.

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(Delta 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(Delta 375-419) homotetramers. Deletion of amino acids 350-419 prevented both GIRK4 homotetramers and GIRK1/GIRK4(Delta 350-419) channels from being delivered to the cell surface.

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 Gbeta gamma -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 Gbeta gamma -activation and failed to identify any similar residues in GIRK1 (18).

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

parallel 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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Abstract
Introduction
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