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J. Biol. Chem., Vol. 281, Issue 40, 30104-30111, October 6, 2006

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Identification of -Aminobutyric Acid Receptor-interacting Factor 1 (TRAK2) as a Trafficking Factor for the K⁺ Channel Kir2.1^*

From the Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, March 15, 2006 , and in revised form, July 28, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To identify proteins that regulate potassium channel activity and expression, we performed functional screening of mammalian cDNA libraries in yeast that express the mammalian K⁺ channel Kir2.1. Growth of Kir2.1-expressing yeast in media with low K⁺ concentration is a function of K⁺ uptake via Kir2.1 channels. Therefore, the host strain was transformed with a human cDNA library, and cDNA clones that rescued growth at low K⁺ concentration were selected. One of these clones was identical to the protein of unknown function isolated previously as -aminobutyric acid receptor-interacting factor 1 (GRIF-1) (Beck, M., Brickley, K., Wilkinson, H., Sharma, S., Smith, M., Chazot, P., Pollard, S., and Stephenson, F. (2002) J. Biol. Chem. 277, 30079-30090). GRIF-1 specifically enhanced Kir2.1-dependent growth in yeast and Kir2.1-mediated ⁸⁶Rb⁺ efflux in HEK293 cells. Quantitative microscopy and flow cytometry analysis of immunolabeled surface Kir2.1 channel showed that GRIF-1 significantly increased the number of Kir2.1 channels in the plasma membrane of COS and HEK293 cells. Physical interaction of Kir2.1 channel and GRIF-1 was demonstrated by co-immunoprecipitation from HEK293 lysates and yeast two-hybrid assay. In vivo association of Kir2.1 and GRIF-1 was demonstrated by co-immunoprecipitation from brain lysate. Yeast two-hybrid assays showed that an N-terminal region of GRIF-1 interacts with a C-terminal region of Kir2.1. These results indicate that GRIF-1 binds to Kir2.1 and facilitates trafficking of this channel to the cell surface.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Electrical activity in excitable tissues largely relies on tight control of K⁺ channel expression. The physiological relevance of changes in K⁺ channel expression has been demonstrated in a variety of normal and pathological states in heart, brain, and pancreas. It has been found that K⁺ channel protein synthesis, assembly, trafficking, and turnover all contribute to the steady state density of K⁺ channels on the cell surface (reviewed in Ref. 1). The molecular mechanisms underlying this regulation are just beginning to emerge.

Mutation studies identified determinants in K⁺ channel proteins that affect their cell surface expression. Although some of these determinants arrest K⁺ channel surface delivery by protein misfolding (2), others encode information on K⁺ channel processing through the secretory pathway (reviewed in Ref. 3). The latter include endoplasmic reticulum retention (4), endo-plasmic reticulum export (5, 6), and Golgi export (7) signals identified in inwardly rectifying K⁺ channels. It is presumed that these determinants interact with their cognate protein trafficking receptors to facilitate delivery of K⁺ channels to the cell surface. For example, current candidates for such partners include the vesicle coat protein COPI and 14-3-3, which responds to arginine-based folding/trafficking signals in Kir channels (8, 9). Further understanding of K⁺ channel trafficking signals critically depends upon identification and analysis of their specific binding partners.

The majority of proteins that govern K⁺ channel surface expression were discovered on the basis of their physical interaction with K⁺ channels. Examples include GM130 (10), calnexin (11), KChAP (12), PSD93 (13), and PSD95 (14), which were identified in yeast two-hybrid system, and MAGUK proteins, which were cloned using proteomics (15). However, a broader scope of K⁺ channel partners may be revealed by functional cloning. This approach can be based on the expression of functional mammalian Kir2.1 channels in yeast; the Kir2.1 channel complements the K⁺ transport defect in mutant yeast lacking the endogenous K⁺ transporters TRK1 and TRK2 and so allows growth at low K⁺ concentrations (16). Thus, Kir2.1-dependent growth of the trk1 trk2 yeast strain at selective K⁺ concentrations can be used for screening for factors that up-regulate Kir2.1-dependent K⁺ uptake. This approach was used for identification of structural (17, 18), functional (19), and trafficking (9) determinants in K⁺ channels. Furthermore, we used Kir2.1 expression in yeast for screening of chemical libraries for K⁺ channel modulators (20). Here, using this system we screened the cDNA library for mammalian proteins to identify the -aminobutyric acid (GABA)³ receptor interacting factor-1 (GRIF-1) (21) as a regulator of Kir2.1 activity. We then show that GRIF-1 is a binding partner of Kir2.1 that facilitates trafficking of this channel to the cell surface in mammalian cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Yeast Strains—Saccharomyces cerevisiae strain SGY1528 with disrupted native K⁺ transporters TRK1 and TRK2 originates from Ref. 16 and has the following genotype: MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 trk1::HIS3 trk::TRP1. The Kir2.1 channel was transformed into SGY1528 strain using a multicopy vector pYMet-Kir2.1 (17), a derivative of pYES (Invitrogen). Kir2.1 is transcribed under control of methionine-repressible promoter. For library screening we used the SGY1528 derivative named iKir2.1 that expresses chromosomally integrated Kir2.1 fused to Met25 promoter and URA3 selective marker. To construct this strain, pYMet-Kir2.1 plasmid was excised of 2-micron DNA by EcoRV digestion, self-ligated, and transformed into SGY1528. Ura⁺ transformants were selected, and Kir2.1 integration was verified using the following criteria: 1) growth on the medium with 2 mM KCl, but not on less then 1 mM KCl; 2) methionine repression of Kir2.1-dependent growth; and 3) stability of the growth rescue after cultivation in nonselective conditions (rich YEPD medium, supplemented to 100 mM KCl). The presence of Kir2.1, URA3, and Met25 promoter after nonselective growth was verified by PCR.

Yeast Cultivation—K⁺-free medium contained 2% glucose, 0.16% yeast nitrogen base without amino acids, sodium, and ammonia, 8 mM H₃PO₄, 2 mM MgSO₄, 0, 2 mM CaCl₂, 0.1% vitamin mix, 0.1% trace elements mix, 0.2% adenine, de-ionized water (adapted from Ref. 16). The media were adjusted to pH 6.5 with arginine base and sterilized by filtering. KCl was added to desired concentration. For preparation of plates with low [K⁺] agarose instead of agar was used. Yeast medium ingredients were from Qbiogene. Yeast transformation was performed using the alkali cation method (Qbiogene). For growth assay, logarithmic cultures with starting density of OD₆₀₀ = 0.05 were incubated at 30 °C in a shaker incubator for 48 h. Culture growth was monitored spectrophotometrically at = 600 nm.

Screening of cDNA Library—Host strain iKir2.1 was transformed with a library of human brain cDNA in pGAD vector (Stratagene). Transformants were selected on medium lacking uracil and leucine supplemented to 100 mM KCl and replica plated on medium without uracil, leucine, and methionine supplemented to 0.5 mM KCl. cDNA clones from growing colonies were extracted and tested for a connection with the Kir2.1 channel.

Yeast Two-hybrid Assay—C-terminal fragments of Kir2.1 were fused to GAL4 activation domain in pGADT7 vector (BD Biosciences Clontech). GRIF-1 fragments were fused to the GAL4 DNA-binding domain on pGBKT7 vector (BD Biosciences Clontech). Kir2.1 and GRIF-1-containing plasmids were co-transformed into YRG-2 strain (Stratagene) of the following genotype: MAT ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3 112 gal4-542 gal80-538 LYS2::UAS_GAL4. Transformants were selected on medium lacking leucine and tryptophan. Growth of transformants on the plates without histidine was interpreted as interaction of the test proteins.

Cell Lines—COS and HEK293 cells were grown in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose and 0.02 glutamine, supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, 100 µg/ml streptomycin at 37 °C and 5% CO₂. Kir2.1 and GRIF cDNA in pCDNA3 vector were transfected into COS and HEK293 cells using Lipofectamine reagent. HEK293 cell line stably expressing Kir2.1 channel tagged with the HA epitope was a gift of Dr. Min Li. Stable HEK293 transfectants were maintained in the presence of 200 µg/ml G418.

⁸⁶Rb⁺ Flux Assay—HEK293 cells stably expressing Kir2.1-HA channels were plated on polylysine-coated plates and transformed with GRIF-containing and control plasmids. 24-48 h after transfection cells were loaded with ⁸⁶Rb⁺ for 4 h by incubation with 1 µCi/ml ⁸⁶RbCl. Prior to ⁸⁶Rb⁺ measurements cells were washed two times with ⁸⁶Rb⁺-free medium. Then medium was collected at 1-40 min time points, and the amount of released ⁸⁶Rb⁺ was measured by scintillation counting. At 40 min, the cells were lysed with 1% SDS, and residual radioactivity in cells was determined.

Quantitative Microscopy—COS cells were co-transfected with Kir2.1 channel tagged with GFP at the N terminus and extracellular HA epitope (GFP-Kir2.1-HA, gift from Dr. Nikolaj Klocker) and GRIF-containing or control plasmids. Twenty-four hours after transfection, live cells were blocked with 10% normal goat serum in PBS (NGS) and incubated with primary rat anti-HA antibody 3F10 (Roche Applied Science) diluted 1:500 in 10% NGS. Following wash with PBS cells were incubated with the goat anti-rat antibody conjugated with Texas Red (Jackson Immunoresearch Labs) diluted 1:1000 in 2% NGS. Incubations with antibodies were performed at room temperature for 30 min; PBS washings were performed three times for 5 min each. The cells were visualized at 40x magnification with inverted microscope (Olympus IX70). The cells were illuminated with 488- and 543-nm light. GFP and Texas Red signals, corrected to background, were quantified in captured images of individual cells using SimplePCI software.

Flow Cytometry—HEK 293 cells stably expressing Kir2.1 channel tagged with extracellular HA epitope were transiently transfected with GRIF-containing or control plasmids. Twenty-four hours after transfection, the cells were washed with PBS and harvested by incubation with 0.5 mM EDTA in PBS for 5-10 min at room temperature. Following wash with Hanks' balanced salt solution supplemented with 5 mM Hepes, pH 7.3, and 2% fetal bovine serum, the cells were incubated with rat anti-HA monoclonal antibody 3F10 diluted 1:500 on ice for 1 h. The cells then were washed twice with Hanks' balanced salt solution and incubated with Cy2-labeled goat anti-rat antibody diluted 1:1000 (Jackson ImmunoResearch) for 15 min on ice. Finally, the cells were washed twice with Hanks' balanced salt solution, and fluorescence was measured by FACSCalibur (Becton Dickinson). The percentage of fluorescent cells was analyzed using CELLQUEST software (Becton Dickinson).

Immunoprecipitation and Western Blot—HEK293 cells were plated in 100-mm culture dish at the density of 3x10⁶/dish and the next day co-transfected with Kir2.1 and FLAG-tagged GRIF constructs. After 24 h of incubation, the cells were washed with PBS and lysed with 500 µl/dish radioimmune precipitation assay buffer (150 mM NaCl, 10 mM Tris, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, 5 mM EDTA, protease inhibitor mixture, pH 7.2) for 30 min at 4 °C. The cell lysates were clarified by centrifugation at 14,000 rpm for 10 min. The supernatants were incubated with 30 µl anti-FLAG resin (Sigma) for 1 h at 4 °C. The supernatants were aspirated, and the pellets were gently rinsed three times with 1 ml of lysis buffer. The immune complexes were eluted with 50 µl of 100 µg/ml FLAG peptide in lysis buffer, mixed with Laemmli buffer, boiled for 10 min, and analyzed by immunoblotting. Following electrophoresis in 10% SDS-polyacrylamide gel proteins were transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk in PBS with 0.5% Tween (PBST) for 1 h at room temperature and incubated overnight at 4 °C with polyclonal anti-Kir2.1 antibody (Alomone) diluted 1:1000. After double washing with PBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody. Western blots were developed using high sensitivity Pierce ECL kit. Complexes of GRIF-FLAG with the Kir2.1 C terminus tagged with Myc were precipitated using anti-FLAG resin as above and probed with anti-Myc antibody (Abcam) diluted 1:3000 for 1 h at 4 °C. The cells transfected with GRIF₄₉₇-X-press and Kir2.1 C terminus tagged with Myc were lysed as above. Clarified lysates were incubated with 2 µl of rabbit polyclonal anti-X-press antibody (Invitrogen) overnight at 4 °C, and immunocomplexes were captured using 50 µl/sample protein A/G Plus-agarose reagent (Santa Cruz Biotechnology) for 2 h at 4 °C. The beads were washed four times with 1 ml of lysis buffer, mixed with Laemmli buffer, and incubated for 10 min at 95 °C for elution. The eluates were subjected to 8% SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with monoclonal anti-Myc antibody (Abcam) diluted 1:3000.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. GRIF clones rescue Kir2.1-dependent yeast growth. A, growth of yeast transformants on medium with 0.5 mM KCl. Sector 1, trk1trk2 knock-out; sector 2, trk1trk2 transformed with pYMetKir2.1; sector 3, trk1trk2 expressing a chromosomal copy of Kir2.1; sector 4, previous strain transformed with KP39 (hGRIF497); sector 5, trk1trk2 transformed with KP39; sector 6, iKir2.1 host transformed with GRIF-1. Transformants in sectors 1-3 carry empty p425 vector with selective marker LEU3, and in sectors 1 and 5, transformants carry empty pYMet with URA3 to support growth on medium without uracil and leucine. B, growth of yeast co-transformed with Kir2.1 and KP39 or empty vector in selective medium with 0.5 mM KCl and nonselective medium with 100 mM KCl. The bars represent the mean optical density (OD600) of three cultures ± S.E. after 48 h of cultivation. C, growth curve of iKir2.1 transformed with KP39, GRIF-1, or vector in medium with 0.5 mM KCl. OD600 was measured at indicated time points.

Immunofluorescent Cell Staining—COS cells were plated on glass cover-slips and transfected with epitope-tagged GRIF-1-FLAG and Kir2.1-HA constructs. Twenty-four hours after transfection the cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature, permeabilized by incubation in 0.5% Triton X-100 in PBS for 15 min at room temperature, and blocked with 20% NGS in PBS for 20 min at room temperature. The cells were incubated with rabbit anti-FLAG antibody (Cell Signaling) diluted 1:400 and mouse anti-HA 12CA5 antibody (Roche Applied Science) in 20% NGS overnight at 4 °C. Following wash with PBS cells were incubated with the secondary goat anti-rabbit FITC-conjugated antibody (Upstate) diluted 1:500 in 5% NGS, washed with PBS, incubated with secondary goat anti-mouse antibody conjugated with Cy3 diluted 1:500 (Jackson Immunoresearch Labs) in 5% NGS, and washed with PBS. Incubations with secondary antibodies were performed for 30 min at room temperature. PBS washes were performed three times for 5 min each at room temperature. The specimens were mounted in mounting medium (Invitrogen) and observed with fluorescent inverted microscope Olympus under 40x magnification in 488- and 543-nm light. No cross-reactivity was detected between 12CA5 antibody and anti-rabbit FITC conjugate or rabbit anti-FLAG antibody and anti-mouse Cy3 conjugate.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Isolation of GRIF-1 as an Enhancer of Kir2.1-dependent Yeast Growth—Growth of a yeast trk1 trk2 double mutant at low K⁺ concentration is rescued by expression of mammalian Kir2.1 channels (Fig. 1A). Kir2.1 expressed from a single chromosomally integrated copy restores growth at 2 mM KCl but not at 0.5 mM KCl (Fig. 1A, sector 3). In contrast, transformation with a 2 micron-based multicopy Kir2.1 plasmid enables growth at 0.5 mM KCl (Fig. 1A, sector 2). Using this dosage effect, we sought to identify mammalian cDNAs that enable single copy Kir2.1-dependent growth at 0.5 mM KCl and thus are likely to encode positive regulators of Kir2.1. The single copy Kir2.1 strain, iKir2.1, was transformed with a human cDNA yeast expression library, and transformants able to grow at 0.5 mM KCl were selected. To confirm cDNA dependence of the growth phenotype, library plasmids were rescued from yeast transformants in Escherichia coli, and their ability to confer yeast growth at 0.5 mM KCl was tested upon retransformation into the iKir2.1 strain. One of the confirmed cDNAs, KP39, promoted growth at 0.5 mM KCl (Fig. 1, A, compare sector 4 with sector 3; and B, left panel). However, KP39 did not increase growth at 0.5 mM KCl of the trk1 trk2 strain in the absence of Kir2.1 (Fig. 1A, sector 5). Likewise, it did not improve growth at a high, nonselective K⁺ concentration (Fig. 1B), a condition in which Kir2.1 is not necessary for growth. Therefore, the KP39-encoded protein does not generally increase growth. Rather, the facilitation of growth by KP39 is coupled to Kir2.1 channel function.

View larger version (12K): [in this window] [in a new window]   FIGURE 2. GRIF clones enhance Kir2. 1-mediated 86Rb+ efflux in HEK293 cells. A, time course of 86Rb+ efflux from stable Kir2.1 cell line transiently transfected with GRIF-1, hGRIF497, and control plasmid. The cells were loaded with 86Rb+ and washed, and 86Rb+ release was measured at the indicated time points. B, amount of 86Rb+ released during the first 3 min after washing from cells co-expressing Kir2.1 with GRIF clones or control vector (vect.). The bars represent the mean release ± S.E. in 22 and 11 independent transfections for GRIF-1 and hGRIF497 samples, respectively. p < 0.001 as determined by paired t test.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. GRIF promotes surface expression of Kir2.1 channel in mammalian cell lines. A, flow cytometry analysis of Kir2.1 surface expression. HEK293 cells stably expressing Kir2.1-HA were transfected with GRIF-1 and hGRIF497. Surface Kir2.1 channels were stained with antibody against extracellular HA epitope and Cy2-conjugated secondary antibody. B, quantification of the flow cytometry results. The bars represent the mean Kir2.1 fluorescence in GRIF-expressing and control groups, corrected for background and normalized to control, ± S.E. n = 5-9 independent transfections. p < 0.05 as determined by paired t test. C, quantitative microscopy of Kir2.1 surface expression. GFP-Kir2.1-HA construct was co-transfected with GRIF-1 or control plasmid into COS cells. Surface expression was measured as signal associated with immunostaining of extracellular HA epitope normalized to GFP fluorescence. The bars represent the mean from >300 cells ± S.E. in each group. D, total cellular GFP-Kir2.1 expression in the presence and absence of GRIF. GFP fluorescence in COS cells expressing GFP-Kir2.1 with GRIF-1 or control plasmid. The bars represent the mean fluorescence measured as in C in 500 cells ± S.E. vect., vector.

GRIF-1 Increases Kir2.1-mediated ⁸⁶Rb⁺ Efflux in Mammalian Cells—To examine effects of GRIF-1 expression on Kir2.1 function in mammalian cells, we used the ⁸⁶Rb⁺ flux assay. Because hydrated Rb⁺ ion is similar in permeability to K⁺, ⁸⁶Rb⁺ flux can be used as an indicator of K⁺ channel activity. HEK293 cells stably expressing Kir2.1 were transiently transfected with GRIF-1 constructs or corresponding empty vectors. The cells were then loaded with ⁸⁶Rb⁺ and washed, and the time course of ⁸⁶Rb⁺ release was determined. In these assays, channel activity is revealed in the initial rate of release. As seen in Fig. 2A, initial efflux of ⁸⁶Rb⁺ from cells transfected with full size GRIF-1 or hGRIF₄₉₇ fragment occurs faster than from vector-transfected cells. Measuring ⁸⁶Rb⁺ efflux from multiple independent transfections proved the statistical significance of this difference (Fig. 2B). Overall, GRIF-1 expression in 40% of the cells increased total Kir2.1-mediated ⁸⁶Rb⁺ flux by 12% ± 1.7%. Thus, channel activity was increased by 30% by GRIF-1. In contrast, transfection of GRIF-1 constructs into HEK293 cells that do not express Kir2.1 had no effect on the background ⁸⁶Rb⁺ efflux, which was 10% of that in Kir2.1-transfected cells (data not shown). Therefore, we conclude that GRIF expression increases Kir2.1 channel activity.

View larger version (47K): [in this window] [in a new window]   FIGURE 4. GRIF-1 is physically associated with the Kir2.1 channel. A, co-immunoprecipitation (IP) of GRIF-1-FLAG and Kir2.1 from HEK293 lysates. Lysates were precipitated on anti-FLAG resin and probed with anti-Kir2.1 antibody. B, co-immunoprecipitation of GRIF-1 with C terminus of Kir2.1. HEK293 cells were co-transfected with Kir2.1 (180-428)-Myc and GRIF-1-FLAG. The lysates were precipitated on anti-FLAG resin and immunoblotted (IB) with anti-Myc antibody. C, co-immunoprecipitation of GRIF-1 N-terminal fragment with C terminus of Kir2.1. Kir2.1 (180-428)-Myc was co-transfected with GRIF (1-497)-X-press. The lysates were precipitated using anti-X-press antibody and immunoblotted with anti-Myc antibody. D, yeast two-hybrid analysis of Kir2.1-GRIF interaction. Yeast strain YRG-2 was co-transformed with C terminus of Kir2.1 fused to GAL4 AD and GRIF-1 and hGRIF497 fused to GAL4 BD. Control fusions SV40-AD and p53-BD interact in yeast two-hybrid assay. Growth of transformants on media without histidine indicates protein-protein interaction. E, co-immunoprecipitation of native brain Kir2 channels and GRIF-1. Detergent-solubilized protein from whole murine brain was precipitated with anti-Kir2.1/Kir2.2 antibody or control preimmune IgG and probed with anti-GRIF-antibody.

View larger version (32K): [in this window] [in a new window]   FIGURE 5. GRIF-1 binding domain localizes to the C terminus of Kir2.1 channel. A, co-immunoprecipitation (IP) of GRIF-1 with fragments of Kir2.1 C terminus. HEK293 cells were transiently transfected with GRIF-1-FLAG and Kir2.1 fragments 265-428 and 298-428 tagged with Myc epitope. The lysates were precipitated on anti-FLAG agarose and immunoblotted (IB) with anti-Myc antibody. B, yeast two-hybrid mapping of GRIF-binding domain in C terminus of Kir2.1. The black lines indicate Kir2.1 fragments that interact with GRIF497. C, sequence of Kir2.1 348-396 fragment required for Kir2.1-GRIF interaction. The coiled coil is underlined, and the trafficking signals are in bold type. The asterisk indicates the predicted O-GlcNAc modification site.

To confirm data obtained using immunoprecipitation, we used the yeast two-hybrid assay. A cytoplasmic domain of Kir2.1 (amino acids 180-428) was inserted into the GAL4 transcription activation domain (AD) vector, and full-length GRIF-1 or its N-terminal half (amino acids 1-497) were inserted into the GAL4 DNA-binding domain (BD) vector. AD and BD constructs were then co-transformed into the YRG-2 yeast strain whose growth in the absence of histidine depends on functional Gal4 transcriptional activator. If Kir2.1 and GRIF-1 associate, this should bring AD and BD portions of Gal4 in close proximity, thus reconstructing functional Gal4 and allowing growth of YRG-2 in the absence of histidine. As shown in Fig. 4D, the cytoplasmic domain of Kir2.1 associates with full-size GRIF-1 as well as with its N-terminal half. This association is similar in strength to that between the positive control pair, SV40-AD and p53-BD. None of the GRIF constructs showed association with SV40-AD. Similarly, the Kir2.1 C terminus fused to AD did not confer histidine-independent growth alone or in combination with p53-BD, demonstrating the specificity of Kir2.1-GRIF-1 association (Fig. 4D). Thus, yeast two-hybrid results confirm biochemical results, and both sets of data indicate specific association of the C-terminal cytoplasmic domain of Kir2.1 with the N-terminal half of GRIF-1.

Finally, to determine whether the Kir2.1-GRIF-1 interaction occurs in vivo, we attempted co-immunoprecipitation of these two proteins from murine brain. Fig. 4E (IP Kir2) shows that precipitate collected with anti-Kir2.1/Kir2.2 antibodies contains GRIF-1 protein. In contrast, control precipitate collected with nonspecific preimmune serum gave no signal (Fig. 4E, mock IP). Thus, native Kir2 and GRIF proteins associate in vivo, indicating the physiologic relevance of the interaction originally discovered with heterologous expression.

Localization of GRIF-binding Segment in Kir2.1—To identify Kir2.1 regions responsible for association with GRIF-1, we constructed deletions of Kir2.1 C terminus (180-428 amino acids) and examined their association with GRIF-1. Co-immunoprecipitation showed that N-terminal truncation of 118 residues in the 180-428 fragment does not disrupt interaction with GRIF-1 (Fig. 5A). Detailed mapping of the GRIF-binding domain in Kir2.1 C terminus (180-428) was then performed using a yeast two-hybrid assay. Truncations removing amino acids 180-348 or amino acids 396-428 preserved the Kir2.1/GRIF₄₉₇ interaction (Fig. 5B). Consistent with these results, the 348-396 region retained the ability to associate with GRIF₄₉₇. However, truncations extending into the 348-396 region further (185-360, 185-375, as well as double truncation 185-330, 381-428) abrogated the yeast two-hybrid interaction. These data indicate that the region of Kir2.1 responsible for association with GRIF₄₉₇ is located between amino acids 348 and 396, with the 360-381 sequence being essential (Fig. 5C).

View larger version (90K): [in this window] [in a new window]   FIGURE 6. Co-localization of GRIF-1 and Kir2.1 in COS cells. A, COS cell transfected with GRIF-1-FLAG and immunostained with mouse anti-FLAG antibody and goat anti-mouse FITC-conjugated secondary antibody. B-D, a representative COS cell co-transfected with GRIF-1-FLAG and Kir2.1-HA and double immunostained for GRIF-1 and Kir2.1. The cells were stained with primary rabbit anti-FLAG and mouse anti-HA antibodies and secondary anti-rabbit FITC conjugate and anti-mouse Cy3 conjugate. B, red Cy3 signal associated with Kir2.1. C, green FITC signal associated with GRIF-1. D, overlay of B and C. The yellow signal indicates areas of Kir2.1 and GRIF-1 co-localization.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Here we report a novel protein partner of the mammalian K⁺ inwardly rectifying channel Kir2.1. This protein is encoded by the ALS2CR3 gene mapping in the human juvenile amylotrophic lateral sclerosis critical region, 2q33-2q34. The rat ortholog of human ALS2CR3 protein, GRIF-1, was identified recently as a binding partner of the -subunit of GABA receptor (21). Based on GRIF-1 binding to kinesin and its homology with proteins implicated in vesicular and organelle motility, a role for GRIF-1 as a trafficking factor has been proposed (23). We identified a direct effect of GRIF-1 to promote K⁺ channel surface expression and activity.

Human and rat GRIF proteins enable growth at low K⁺ concentration of a trk1 trk2 yeast strain expressing a single copy of Kir2.1. Enhancement by GRIF-1 of Kir2.1-dependent K⁺ flow is not a yeast-specific phenomenon because similar enhancement occurs in mammalian cells (HEK293), as indicated by ⁸⁶Rb⁺ flux data. In mammalian cells, ectopic expression of full-length or the N-terminal half of GRIF-1 increases surface expression but not total Kir2.1 protein expression. This suggests that GRIF-1 increases K⁺ currents via increased delivery of Kir2.1 to the plasma membrane. Kir2.1 and GRIF-1 not only functionally interact, they also co-precipitate from mammalian cell lysates, associate in the yeast two-hybrid assay, and co-localize when expressed in mammalian cells, all indicating specific physical association. Furthermore, co-immunoprecipitation from brain indicates that native Kir2.1 and GRIF-1 proteins associate in vivo. The site responsible for association with GRIF-1 is located in the cytoplasmic tail of Kir2.1 between residues 348 and 396. This 49-amino acid-long region contains signals critically important for Kir2.1 trafficking from endo-plasmic reticulum to plasma membrane, as well as putative coiled coil, amphipatic helix, and O-GlcNAc modification sites. Based on these findings, we propose that GRIF-1 acts as a binding partner of Kir2.1 that facilitates trafficking of this K⁺ channel through the secretory pathway.

GRIF-1 belongs to the TRAK family of coiled coil domain-containing proteins implicated in trafficking of vesicles and mitochondria. The family includes the O-GlcNAc transferase-interacting protein OIP (24); Milton, a protein transporting mitochondria to neuronal synapses in Drosophila (25); and TRAK1, a protein required for GABA receptor homeostasis in mice (26). All of these proteins specifically bind the heavy chain of the microtubule-associated motor protein kinesin (23, 25). It has been proposed that GRIF/TRAK proteins provide a link between specific cargo proteins or organelles and kinesin motors (23, 25, 26). A trafficking adaptor role for GRIFs finds further support in the fact that the N-terminal half of GRIF-1 contains a stretch of 300 amino acids that is 47% homologous to the huntingtin-associated protein HAP-1 (27), which plays a role in trafficking of proteins and organelles in neurons by linking them to kinesin (28). Therefore, it is possible that GRIF-1 (TRAK2) links Kir2.1 to kinesin heavy chain, which is important for delivery of K⁺ channels to the cell surface (29).

The role of GRIF-1 as a trafficking factor for Kir2.1 is also supported by its binding to a segment of Kir2.1 that contains structural features relevant for protein-protein interaction and Kir2.1 trafficking signals (Fig. 5C). First, the 350-380 region of Kir2.1 contains regularly spaced hydrophobic amino acids that may form an amphipatic -helix, and the 376-389 region is predicted to assume a coiled coil structure (29). A coiled coil structure is also located in the N-terminal half of GRIF-1 and is essential for interaction with GABA receptor (21). Thus, it is possible that coiled coil domain in Kir2.1 interacts with a similar structure in the N-terminal half of GRIF-1 and so contributes to Kir2.1-GRIF-1 association. Second, threonine-353 in the GRIF-binding region of Kir2.1 is a putative site of O-Glc-NAc modification (30). The addition of O-GlcNAc to serine or threonine residues is a labile modification that plays signaling role and may be an alternative to phosphorylation at the same site (reviewed in Ref. 31). Several proteins known to be modified by O-GlcNAc have functions that are relevant to the secretory pathway, including Golgi reassembly stacking protein 2 (GRASP55), cytoskeletal proteins, and microtubule assembly proteins MAP1B and MAP2B (32). This suggests that O-Glc-Nac signaling plays a role in protein trafficking. In light of the reported association of GRIF-1 with O-GlcNAc-transferase (24), it cannot be excluded that GRIF-1 facilitates the addition of O-GlcNac to Kir2.1 and thus provides a dynamic signal for the channel trafficking. Finally, the GRIF-1-binding segment of Kir2.1 contains at least two signals required for the efficient trafficking of this K⁺ channel to the plasma membrane. These signals, FCYENE and EEEEDSE, direct export from the endo-plasmic reticulum and post-Golgi vesicles, respectively, and therefore are likely binding sites for distinct trafficking factors (6, 7). FCYENE and EEEEDSE are necessary, but not sufficient for Kir2.1 surface expression because a larger segment (amino acids 369-410), which incorporates both signals and overlaps with GRIF-1-binding site (amino acids 348-396), is required to confer surface expression in a domain swap assay (6). Because FCYENE and EEEEDSE are parts of the essential GRIF-1-binding segment, it is possible that GRIF-1 plays a role in recognition of these signals. If GRIF-1 is a trafficking factor for Kir2.1, it is likely to act in concert with other factors that bind to the cytoplasmic tail of Kir2.1, such as actin-binding protein filamin A (33), vesicle coat proteins adaptins (34), or proteins with PDZ motifs (13, 15). Preferential expression of GRIF-1 in heart, brain, and skeletal muscle (21) is also consistent with its role as a trafficking partner for Kir2.1, which is specifically expressed in these tissues.

	FOOTNOTES

 
^* This work was supported by a beginning grant-in-aid award from the American Heart Association (to E. Z.-M.) and National Institutes of Health Grants HL80632 and HL55312 (to E. S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Division of Pediatric Surgery, Children's Hospital of Los Angeles, Los Angeles, CA 90027.

² To whom correspondence should be addressed: Dept. of Pharmacology, University of Pittsburgh School of Medicine, 220 Lothrop St., Pittsburgh, PA15261. Tel.: 412-648-9486; Fax: 412-648-1945; E-mail: levitan{at}server.pharm.pitt.edu.

³ The abbreviations used are: GABA, -aminobutyric acid; GRIF, -aminobutyric acid receptor-interacting factor; HA, hemagglutinin; GFP, green fluorescent protein; PBS, phosphate-buffered saline; NGS, normal goat serum; FITC, fluorescein isothiocyanate; AD, activation domain; BD, DNA-binding domain; TRAK2, trafficking protein, kinesin binding 2.

	ACKNOWLEDGMENTS

 
We thank Dr. Anne Stephenson for GRIF-1 clone and GRIF antibody, Dr. Nikolaj Klocker for the GFP-Kir2.1-HA construct, Dr. Min Li for the HEK293 Kir2.1-HA stable cell line, and Dr. Carol Vandenberg for Kir2.1 antibody.

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