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Originally published In Press as doi:10.1074/jbc.M707738200 on February 26, 2008

J. Biol. Chem., Vol. 283, Issue 18, 12241-12247, May 2, 2008
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MAGI-1a Functions as a Scaffolding Protein for the Distal Renal Tubular Basolateral K+ Channels*Formula

Masayuki Tanemoto1, Takafumi Toyohara, Takaaki Abe, and Sadayoshi Ito

From the Division of Nephrology, Hypertension, and Endocrinology, Department of Medicine, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-857, Japan

Received for publication, September 14, 2007 , and in revised form, February 7, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
As the K+ recycling pathway for renal Na+ reabsorption, renal tubular K+ channels participate in the fluid and electrolyte homeostasis. Previously, we showed that the Kir5.1/Kir4.1 heteromer, which is a heteromeric assembly of two inwardly rectifying K+ channels, composes the principal basolateral K+ channels in distal renal tubules and that two motifs in the carboxyl-terminal portion of the Kir4.1 subunit regulate its functional expression. In this study, by using yeast two-hybrid screening, we identified a new isoform of membrane-associated guanylate kinase with inverted domain structure 1 (MAGI-1a-long) as a scaffolding protein for the basolateral K+ channels. MAGI-1a-long interacted with the PSD-95/Dlg/ZO-1 (PDZ)-binding motif of Kir4.1 by its fifth PDZ domain, and a high salt diet, which could suppress mineralocorticoid secretion, facilitated the interaction. The phosphorylation of serine 377 in the PDZ-binding motif disrupted the interaction, and the disruption of the interaction altered the intracellular localization of the channels from the basolateral side to perinuclear components. These results demonstrate that the phosphorylation-dependent scaffolding of the basolateral K+ channels by MAGI-1a-long participates in the renal regulation of the fluid and electrolyte homeostasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Kidney is the essential organ for the fluid and electrolyte homeostasis, and the derangement of its function results in life-threatening diseases, including hypertension, the most common disease in industrialized societies (1–3). The Mendelian form abnormalities have provided clues to pathophysiology of many diseases, and recent genetic analysis revealed that the Na+ reabsorption in distal renal tubules participates in the pathogenesis of hypertension (4, 5).

The process of the Na+ reabsorption in distal renal tubules is the result of coordinated electrolyte transports across the renal epithelia (5, 6). In the epithelia, Na+ efflux occurs actively via basolateral Na+/K+-ATPases, whereas apical pathways passively mediate Na+ influx. Different pathways mediate the apical Na+ influx in each segment of the tubules as follows: Na+-K+-2Cl- cotransporters in the thick ascending limbs of Henle's loop (TAL),2 Na+-Cl- cotransporters in the distal convoluted tubules (DCT), and Na+ channels in the connecting tubules and the collecting ducts. Because Na+/K+-ATPases induce intracellular K+ influx with simultaneous Na+ efflux in these segments, K+ excretion, i.e. K+ recycling, is essential for the continuance of Na+ reabsorption (7).

Previously, we showed that the Kir5.1/Kir4.1 heteromer, which is a heteromeric assembly of two inwardly rectifying K+ channels, composes the principal pathway for the basolateral K+ recycling in the distal portion of TAL and the DCT (8–10). The functional expression of this heteromer is regulated by the following two motifs in the carboxyl-terminal (CT) portion of the Kir4.1 subunit: (i) dihydrophobic motif for functional cell-surface expression and (ii) PSD-95/Dlg/ZO-1 (PDZ)-binding motif for basolateral localization (11). Therefore, the renal tubules are believed to regulate the basolateral K+ recycling by the mechanism that recognizes these motifs, and the mechanism is a key factor for the renal regulation of Na+ and K+ homeostasis (6, 12).

This study reports that an isoform of membrane-associated guanylate kinase with inverted domain structure 1 (MAGI-1a) functions as a scaffolding protein for the channels that compose the basolateral K+ recycling pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Two-hybrid Screening and Cloning—The CT of rat Kir4.1, which is a fragment of 105 amino acid residues, was fused to a GAL4 DNA-binding domain, and 500,000 clones of a rat kidney cDNA library were screened by using the Matchmaker GAL4 two-hybrid system (Takara Bio Clontech) according to the manufacturer's instructions. The sequence of several clones showed high homology to the sequence of the CT domain of mouse MAGI-1a. The full-length of rat MAGI-1a isoforms, MAGI-1a-long and MAGI-1a-short, was cloned by PCR from rat kidney cDNA with the primer pairs of 5'-ATGTCGAAAGTGATCCAGAA-3' and 5'-TCATGGAGTCATGCCAGGGAAGG-3'. For expression analysis of MAGI-1a isoforms in rat tissues, we performed PCR on Rat MTC Panel I (Clontech) using the primer pairs of 5'-ATGTCGAAAGTGATCCAGAA-3' and 5'-CCGAGGGTCTAACCATGATG-3'.

Construct of Fusion Proteins and Deletion/Point Mutants—Mammalian cell expression vectors that contain green fluorescent protein (GFP)-tagged Kir4.1 (GFP-Kir4.1), its deletion/point mutants, and MAGI-1a isoforms were constructed as described previously (11). Polyhistidine-tagged CT81 segments with and without mutation were constructed by subcloning PCR-amplified DNA fragments into the expression vector pcDNA4/HisMaxC (Invitrogen).

Transient Expression in Mammalian Cell Lines—Human embryonic kidney (HEK)293T cells and Madin-Darby canine kidney (MDCK) cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (both from Invitrogen) and were transfected with the expression vectors by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. MDCK cells were plated on polycarbonate Millicell transwell filters (Millipore, Bedford, MA) prior to transfection. The further analysis of expressed proteins was usually conducted at 48–72 h after transfection as described previously (13).

Antibodies—A polyclonal anti-MAGI-1 antibody was raised in rabbits against the synthetic peptide SFTADSGDQDEPTLQEATL that corresponds to amino acids 258–267 of MAGI-1a-long (amino acids 39–58 of MAGI-1a-short). A polyclonal anti-K+-channel Kir4.1 antibody (Sigma), BD Living ColorsTM Av peptide antibodies (Takara Bio Clontech), Alexa Fluor 594 anti-rabbit IgG (Fab')2 (Molecular Probes, Eugene, OR), and fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG (Dako, Glostrup, Denmark) were purchased.

Protein Precipitation and Immunoblotting Analysis—Protein precipitation and immunoblotting analysis were performed as described previously (8). The antibody-pretreated protein A-Sepharose (Amersham Biosciences) was used for immunoprecipitation. The protein A-Sepharose pretreated with preimmune rabbit IgG was used for negative control. Ni-NTA-agarose (Qiagen, Hilden, Germany) was used for polyhistidine-tagged fragments precipitation. The precipitated proteins were analyzed by immunoblotting and detected by SuperSignal West Dura Extended Duration Substrate (Pierce). The intensity of the detected bands was calculated using NIH Image software (National Institutes of Health, Bethesda).

Immunohistochemical Analysis—Adult Sprague-Dawley rats were anesthetized in accordance with the regulations of the Animal Care Committee of our institute, and then perfused transcardially with 0.9% saline followed by perfusion with 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.4). After dehydration, the kidney was embedded in paraffin, and thin sections of 3–5-µm thickness were obtained. After deparaffinization and rehydration, the sections were immunostained and then observed using a confocal microscope (model LSM 5 PASCAL, Carl Zeiss Co., Ltd., Jena, Germany) as described previously (10). In brief, after incubation in blocking buffer containing 5% normal goat serum, the sections were incubated with the anti-Kir4.1 antibody followed by incubation with an excess (1:20 dilution) of Alexa Fluor 594 anti-rabbit IgG (Fab')2 to saturate the epitopes of the primary antibody. After being washed extensively with phosphate-buffered saline, the sections were subsequently incubated with the anti-MAGI-1 antibody followed by incubation with FITC-labeled anti-rabbit IgG at 1:100 dilution. The sections were observed by a confocal microscope after extensive washing with phosphate-buffered saline. The saturation of the first primary antibody was confirmed by the preliminary experiment that did not detect any labeling with FITC by subsequent incubation with control rabbit IgG.

Salt Loading for Animals—At 8 weeks of age, the diet of rats was switched from a 0.3% NaCl to an 8% NaCl (high salt)-containing diet for 2 weeks. Control rats were fed with a 0.3% NaCl diet throughout the experiments.

Glutathione S-Transferase (GST) Pulldown Analysis—GST fusion proteins of several CT segments of MAGI-1a were constructed by subcloning PCR-amplified DNA fragments into the bacterial expression vector pGEX-5X3 (Amersham Biosciences), and each fusion protein was purified on glutathione-SepharoseTM 4B (Amersham Biosciences) according to the manufacturer's protocol. GFP-Kir4.1 resolved as described above was incubated with these purified GST fusion proteins and then analyzed by immunoblotting as described previously (13).

Protein Kinase A (PKA) Phosphorylation—Experiments involving PKA stimulation were performed using a cAMP mixture containing 5 µM forskolin, 100 µM 8-Br-cAMP, and 100 µM 3-isobutyl-1-methylxanthine. The cAMP mixture was applied for 10 min until just before further preparation. For the inhibition of PKA, a PKA-specific blocker, N-(2([3-(4-bromophenyl)-2-propenyl]amino)-ethyl)-5-iso-quinolinesulfonamide (H89) (Seikagaku Corp., Tokyo, Japan) was applied at a concentration of 1.0 µM from 3 min prior the application of the mixture (14).

Electrophysiological Recordings—Channel activity was analyzed with the patch clamp method in whole-cell configuration as described previously (8). In brief, the resistance of patch electrodes, when filled with the pipette solution (140 mM KCl, 1 mM MgCl2, 1 mM CaCl2 and 5 mM HEPES-KOH (pH 7.4)) was adjusted to 1–2 megohms. Currents were elicited on GFP-Kir4.1-transfected HEK293T cells by voltage steps in the bath solution containing 120 mM NaCl, 20 mM KCl, 5 mM EGTA, 2 mM MgCl2, and 5 mM HEPES-KOH (pH 7.3) using a patch clamp amplifier (Axopatch 200B, Axon Instruments, Union City, CA). Data were analyzed by pCLAMP 9 (Axon Instruments), and barium (3 mM)-sensitive components of the currents were recorded as K+ currents.

Reverse Transcription (RT)-PCR Analysis—Total RNA was extracted from MDCK cells using TRIzol reagent (Invitrogen) and reverse-transcribed with (dT)12–18 primer by using Superscript II RT (Invitrogen). MAGI-1a expression was detected by PCR using a primer pair 5'-CCAGTAATTGGGAAATCACACC-3' and 5'-CCGCCTCAGAAACAGACGGAC-3', which spans introns to distinguish these sequences from the genomic DNA. The PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide.


Figure 1
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  		FIGURE 1.
MAGI-1a isoforms expressed in rat kidney. A, exons of MAGI-1 deduced from the sequence in a genomic data base. Arrangements of exons in MAGI-1a isoforms are schematically shown. Splice sites identified in this study are indicated in gray. B, putative domain structures of MAGI-1a-long and MAGI-1a-short. C, expression of MAGI-1a isoforms in rat tissues examined by RT-PCR. Arrowheads in the upper panel indicate the expected sizes for MAGI-1a-long (upper band) and MAGI-1a-short (lower band). Arrowhead in the lower panel indicates the expected sizes for β-actin. NT, amino terminus.

 
Cytological Observation—Cytological observation was performed as described previously (11). Especially in experiments with MDCK cells, formation of tight junction between cells after confluent growth was confirmed by the expression of a typical protein of tight junction, ZO-1, on the top of a lateral wall. Immunostaining was performed in phosphate-buffered saline containing 0.05% Triton X-100, 5% bovine serum albumin, and 1% normal goat serum.

Statistical Analysis—The intensity of the bands in immunoblotting analysis was expressed as means ± S.D. Comparison was performed by the paired two-tailed Student's t test. Probability values of p < 0.01 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of MAGI-1a as a Scaffolding Protein for Kir4.1 in the Kidney—Two isoforms of MAGI-1a were identified from rat kidney cDNA (supplemental Fig. S1). The longer one, named MAGI-1a-long (GenBankTM accession number AY598952 [GenBank] ), had two short inserts to the reported MAGI-1a (before and after the second WW domain). The shorter one, named MAGI-1a-short (GenBankTM accession number AY598951 [GenBank] ), had two splice-out portions from MAGI-1a-long (in the amino terminus and between PDZ2 and PDZ3) (Fig. 1A and supplemental Fig. S1). As a consequence of the splice-out, MAGI-1a-short lacked the amino-terminal PDZ domain (so-called PDZ0) and the guanylate kinase domain (Fig. 1B). Analysis by PCR on rat tissue cDNA revealed that the kidney expressed both MAGI-1a-long and MAGI-1a-short, and MAGI-1a-short was expressed only in the kidney among the tissues examined (Fig. 1C and supplemental Fig. S2).

Expression of MAGI-1a-long-Kir4.1 Complex in Renal Distal Tubules—Using the anti-MAGI antibody, which specifically recognized MAGI-1a-short and MAGI-1a-long expressed in HEK293T cells (Fig. 2A), we confirmed the expression of both MAGI-1a isoforms in the kidney (Fig. 2B). The antibody detected two bands in the lysate from the membrane fraction of rat kidney (Fig. 2B, left panel), the identical size of MAGI-1a-short (lower band) and the predicted size of MAGI-1a-long (upper band). The antibody pre-absorption by the antigenic peptide reduced the detection and almost eliminated these bands (Fig. 2B, right panel).


Figure 2
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  		FIGURE 2.
Intrarenal interaction of MAGI-1a-long and Kir4.1. A, immunoreactivity of the anti-MAGI-1 antibody raised in this study. The antibody specifically detected MAGI-1a isoforms expressed in HEK293T cells. Arrowheads indicate the sizes for MAGI-1a-short (lower band) and MAGI-1a-long (upper band). A representative result of immunoblotting is shown (n = 3). Mock, Kir4.1, short, and long indicate cell lysates from HEK293T cells transfected with mock vector, Kir4.1, MAGI-1a-short, and MAGI-1a-long, respectively. B, renal expression of MAGI-1a isoforms examined by immunoblotting with anti-MAGI-1 antibody. Both MAGI-1a-long (upper arrowhead) and MAGI-1a-short (lower arrowhead) were expressed in rat kidney. The pre-absorption by the antigenic peptide specifically reduced the detection by the antibody (right panel). Representative results of immunoblotting are shown (n = 3 for each). MAGI, lysate from MAGI-1a-short transfected HEK293T cells; Kidney, lysate from rat kidney. C, mutual immunoprecipitation of MAGI-1a-long and Kir4.1 from rat kidney lysate. Immunoprecipitants of preimmune rabbit IgG (Cont), anti-Kir4.1 antibody (anti-Kir4.1), and anti-MAGI-1 antibody (anti-MAGI) were detected by the antibodies indicated. Arrowheads indicate the predicted size of MAGI-1a-long (left panel) and Kir4.1 (right panel). Representative results of immunoblotting (IB) are shown (n = 3 for each).

 
Using the antibody, we next examined the intrarenal interaction between MAGI-1a isoforms and Kir4.1. In the immunoprecipitant with the anti-Kir4.1 antibody, a single band was detected from the rat kidney lysate by the anti-MAGI-1 antibody (left panel in Fig. 2C). The size of the band indicated that the isoform coprecipitated with Kir4.1 was MAGI-1a-long. The immunoprecipitant with the anti-MAGI-1 antibody also contained Kir4.1 (right panel in Fig. 2C). The mutual coprecipitation of MAGI-1a-long and Kir4.1 indicated their intrarenal interaction.

The intrarenal MAGI-1a-long/Kir4.1 interaction was further supported by immunohistochemical analysis (Fig. 3A). The immunoreactivity against the anti-MAGI-1 antibody was detected in the tubules that expressed Kir4.1, and coimmunostaining with the anti-Kir4.1 antibody showed the colocalization of MAGI-1a isoforms with Kir4.1 on the basolateral side in these tubules. The expression of MAGI-1a isoforms on the basolateral side was confirmed by the single staining with the anti-MAGI-1 antibody (supplemental Fig. S3).


Figure 3
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  		FIGURE 3.
Coexpression of MAGI-1a-long/Kir4.1 in rat kidney and regulation of their interaction by dietary amount of NaCl. A, coimmunostaining of rat kidney with the anti-MAGI-1 antibody (green) and the anti-Kir4.1 antibody (red). A representative result of immunostaining is shown (n = 3). MAGI-1a was colocalized with Kir4.1 on the basolateral side of distal tubules in the cortex. Scale bar, 50 µm. B, increased intrarenal MAGI-1a-long/Kir4.1 interaction in the salt-loaded rats. Left and middle panels show representative results of immunoblotting (IB) (n = 4 for each). More Kir4.1 was coprecipitated with MAGI-1a in the kidney of the salt-loaded rat than the control, although nearly the same amount of Kir4.1 is expressed in both (left panels). The antibody used for coprecipitation precipitated a same amount of MAGI-I isoforms in the control and salt-loaded rats (middle panel). Bar graph shows the relative intensity of the coprecipitated Kir4.1 in the salt-loaded rats to that in the control (n = 4; *, p < 0.01). Cont, control; Salt, salt load; IP, immunoprecipitation.

 
Effects of High Salt Diet on Intrarenal MAGI-1a-long/Kir4.1 Interaction—We next examined the effect of high salt diet (salt load), which could suppress mineralocorticoid secretion, on the intrarenal MAGI-1a-long/Kir4.1 interaction (Fig. 3B). The salt load increased the interaction, although it did not increase the amount of Kir4.1 expressed in the kidney. More Kir4.1 was coprecipitated with the same amount of MAGI-1a-long in the kidney of the salt-loaded animals than the control (relative amount: 1.65 ± 0.17, p = 0.005).

Interacting Domains of MAGI-1a-long and Kir4.1—Using GST pulldown, we analyzed the regions for the MAGI-1a-long/Kir4.1 interaction. The CT domain of MAGI-1a-long that was identified by the yeast two-hybrid screening could interact with Kir4.1 (Fig. 4A). Sequential deletion of the domain from the CT end showed that the deletion of the fifth PDZ domain (PDZ5) disrupted the interaction, which indicates that the PDZ5 domain is essential for MAGI-1a-long to interact with Kir4.1.

The deletion of the PDZ-binding motif in Kir4.1 disrupted the MAGI-1a-long/Kir4.1 interaction (Fig. 4B). A single mutation of serine 377, the critical residue in the motif, to alanine (S377A) or aspartic acid (S377D) also disrupted the interaction. These results indicated that the PDZ-binding motif of Kir4.1 was essential for the MAGI-1a-long/Kir4.1 interaction and confirmed that the interaction was PDZ-dependent.

Regulation of the MAGI-1a-long/Kir4.1 Interaction—The PDZ dependence of the MAGI-1a-long/Kir4.1 interaction was further confirmed in HEK293T cells. In HEK293T cells, as well as in the GST pulldown, the Kir4.1 with wild-type (WT) CT portion could interact with MAGI-1a-long, but the deletion of the PDZ-binding motif in Kir4.1 disrupted the mutual interaction between MAGI-1a-long and Kir4.1 (Fig. 5A).


Figure 4
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  		FIGURE 4.
GST pulldown analysis for interacting domains of MAGI-1a-long and Kir4.1. A, GST pulldown of GFP-Kir4.1 by CT segments of MAGI-1a. Schema shows each GST fusion construct and summarizes its binding to Kir4.1. Lower panel shows a representative result of GST pulldown (n = 3). Fusion proteins that contain PDZ5 interacted with Kir4.1; however, deletion of PDZ5 diminished the interaction. B, GST pulldown analysis of GFP-Kir4.1 mutants by PDZ3'-CT segment of MAGI-1a. Schema shows the sequence of CT of each GFP-Kir4.1 mutant and summarizes its binding to MAGI-1a-long. Panels show representative results of expression (upper panel) and pulldown analysis (lower panel) (n = 3). Deletion ({Delta}3) and single mutations (S377A, S377D) in the PDZ-binding motif disrupted the interaction.

 


Figure 5
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  		FIGURE 5.
Effect of PKA phosphorylation on MAGI-1a-long/Kir4.1 interaction. A, coprecipitation of MAGI-1a-long and GFP-Kir4.1 in HEK293T cells. Panels show representative results of immunoprecipitation (by anti-GFP antibody (upper panel) and anti-MAGI-1 antibody (lower panel)). Mutual interaction of Kir4.1 (wild-type, WT) and MAGI-1a-long was disrupted by deletion of the PDZ-binding motif ({Delta}3) from Kir4.1. IB, immunoblot. B, disruption of the MAGI-1a-long/Kir4.1 interaction by PKA phosphorylation. Panel shows a representative result of immunoprecipitation of Kir4.1 by anti-MAGI-1 antibody. Bar graph shows the relative intensity of the Kir4.1 coprecipitated with MAGI-1a-long under PKA stimulation compared with under co-inhibition by H89 (n = 3; *, p < 0.01). The conditions of incubation are indicated in the panel above. The disruption was impeded by H89, a PKA-specific inhibitor. PKA, incubation with cAMP mixture; H89, co-inhibition by H89. C, effect of PKA phosphorylation on Kir4.1 channel activity. The graph summarizes the relative K+ current of Kir4.1 under conditions indicated above the graph (n = 3). No reduction of the channel activity was observed during PKA phosphorylation. CAMP cocktail, cAMP mixture in bath solution; Ba2+, 3 mM barium in bath solution.

 
In the GST pulldown analysis, the induction of a phosphorylated state-mimicking mutation in serine 377 (S377D) disrupted the MAGI-1a-long/Kir4.1 interaction, which indicated that serine 377 phosphorylation affected the interaction. Because serine 377 of Kir4.1 is a putative site for phosphorylation by PKA, we further examined the effects of PKA phosphorylation on the MAGI-1a-long/Kir4.1 interaction in HEK293T cells (Fig. 5B). The interaction was disrupted during 10 min of incubation with the cAMP mixture, whereas a PKA-specific inhibitor, H89, overcame this disruption. Compared with the amount of Kir4.1 coprecipitated with MAGI-1a-long after simultaneous inhibition with H89, the amount after PKA stimulation was significantly low (0.13 ± 0.03, p < 0.001).


Figure 6
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  		FIGURE 6.
Phosphorylation of serine residue in PDZ-binding motif of Kir4.1. A, PKA phosphorylation of CT portion of Kir4.1. Ni-NTA-agarose-precipitated polyhistidine-tagged CT81 segment of Kir4.1 expressed in HEK293T cells was detected by antibodies indicated. His, anti-polyhistidine antibody; P-Serine, anti-phosphoserine antibody. Sequence of segments and condition of treatments are indicated above panels. S377A, mutant with serine 377 to alanine; PKA, stimulation with cAMP mixture; H89, inhibition by H89. Representative immunoblots (IB) are shown in upper panels, and relative intensity is summarized in the bar graph (mean ± S.D., n = 4; *, p < 0.01 compared with WT/PKA+/H89-). B, schematic summary of MAGI-1a-long/Kir4.1 interaction. Only PDZ-binding motif of Kir4.1 with unphosphorylated serine 377 can bind to PDZ5 of MAGI-1a-long.

 
Interestingly, the current amplitude of Kir4.1 assessed by patch clamp did not change during the 10-min cAMP mixture application, which sufficiently disrupted the PDZ-dependent interaction with MAGI-1a-long; the amplitude after the 10-min application relative to that before the application was 0.96 ± 0.05 (Fig. 5C).

Phosphorylation of Serine 377 in the PDZ-binding Motif—We further examined PKA phosphorylation on the CT81 amino acid residues of Kir4.1 (CT81 segment) (Fig. 6A). Whereas Ni-NTA-agarose precipitated the same amount of the WT and S377A polyhistidine-tagged CT81 segments, the WT segment instead of the S377A segment was more efficiently serine-phosphorylated by the cAMP mixture application in the H89-inhibitory manner. Compared with the intensity of phosphoserine on the WT segment, the intensity on the S377A segment was statistically significantly weak (0.25 ± 0.19, p = 0.004), and the reduction in the intensity by simultaneous H89 inhibition was significant only in the WT segment (WT, 0.29 ± 0.05, p < 0.001; S377A, 0.15 ± 0.05, p = 0.305). These results indicated that the serine 377 in the PDZ-binding motif was the residue that was PKA-phosphorylated. The motif with unphosphorylated but not phosphorylated serine 377 could interact with the PDZ5 of MAGI-1a (schematically summarized in Fig. 6B).

In the absence of extra activation for PKA, the intensity of phosphoserine on the WT segment changed widely in each experiment (ranging from 0.33 to 1.13). However, the intensity without the extra activation was generally weaker than the intensity with the extra activation (0.72 ± 0.34, p = 0.201).


Figure 7
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  		FIGURE 7.
PDZ dependence of K+ channel localization in MDCK cells. A, expression of MAGI-1 isoforms in MDCK cells. RT-PCR analysis for the expression of MAGI-1 in MDCK cells by using a MAGI-1-specific primer pair. A representative result of the analysis is shown (n = 3). Lane 1, cDNA of MDCK cells; lane 2, RT(-) total RNA of MDCK cells; lane 3, cDNA of MAGI-1a-long as a positive control. B, intracellular localization of GFP-Kir4.1 mutants in MDCK cells. A representative result of the localization is shown (n = 5). Whereas wild-type (WT) GFP-Kir4.1 is predominantly localized on basolateral side, single phosphorylation-mimic mutation (serine 377 to aspartic acid; S377D) induced perinuclear localization. Scale bar, 10 µm.

 
Effect of Kir4.1 Phosphorylation on Intracellular Localization—We further examined the effect of phosphorylation on the intracellular localization of Kir4.1 in renal tubular cells by using a cell line derived from the renal tubule, MDCK cells. RT-PCR analysis by using a specific primer pair for MAGI-1 revealed the expression of MAGI-1 isoform(s) in MDCK cells (Fig. 7A). In MDCK cells, Kir4.1 showed the predominant localization on the basolateral side, but the induction of a phosphorylation-mimicking single mutation at serine 377 (S377D) induced the perinuclear localization (Fig. 7B). These results indicated that the PDZ-dependent interaction with MAGI-1 isoform(s), which is disrupted by the phosphorylation of serine 377 in the PDZ-binding motif, participated in the basolateral localization of Kir4.1 in renal tubular cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we revealed that a scaffolding protein, MAGI-1a-long, interacts with Kir4.1 on the basolateral side of distal renal tubules. The interaction is affected by dietary amount of NaCl and regulated by phosphorylation of the PDZ-binding motif of Kir4.1.

Previously, we showed that Kir4.1 is a subunit of the basolateral K+ channels (a pathway of basolateral K+ recycling for Na+ reabsorption) in distal renal tubules (8, 9), and that the PDZ-binding motif of this subunit participates in the proper expression of the channels (10, 11). Therefore, our findings in this study indicate that MAGI-1a-long participates in the basolateral localization of the K+ channels in these tubules; this is similar to the PDZ-dependent localization of other membrane proteins by scaffolding proteins (15).

We detected the expression of MAGI-1 isoforms in the renal tubules but not in the glomerulus. The intrarenal interaction of MAGI-1a-long with Kir4.1, which is not expressed in the glomerulus but in distal renal tubules (10, 16), confirmed the tubular expression of MAGI-1 isoforms. In a previous report, another MAGI-1-specific antibody also detected tubular but not intraglomerular expression of MAGI-1 (17). However, there are several reports that show intraglomerular expression of MAGI-1 (18–20). In these previous reports, the antibodies that were raised against other members of scaffolding proteins (19, 20) or that recognized several proteins with sizes different from MAGI-1 (18) were used, because they could detect MAGI-1. Therefore, intraglomerular immunoreactivity with these antibodies could be against some other proteins, such as a synaptic scaffolding molecule, but not MAGI-1 (21). It is also possible that isoform(s) of MAGI-1 without the amino-terminal portion are expressed in the glomerulus, because we raised the anti-MAGI-1 antibody against an amino acid sequence on the amino-terminal portion of MAGI-1a in this study.

In the renal tubules, the MAGI-1 expression was detected on the luminal side in addition to the basolateral side where MAGI-1a-long was colocalized with Kir4.1. Because MAGI-1a-short is the renal MAGI-1 isoform that did not interact with Kir4.1, it would be the isoform expressed on the luminal side. Although we could not clarify the precise localization of the isoforms, they probably have different intracellular localizations, i.e. MAGI-1a-short on the luminal side and MAGI-1a-long on the basolateral side. The PDZ0 and guanylate kinase domain, the domains that only MAGI-1a-long contains, might be responsible for the different localization (15). The different localization would make it possible for MAGI-1a-long but not MAGI-1a-short to interact with Kir4.1 in vivo in the kidney.

The mechanism for in vivo interaction of Kir4.1 with the basolaterally expressed MAGI-1a-long but not the luminally expressed MAGI-1 isoforms has not been clarified. However, we propose two possible mechanisms. (i) PDZ5 of the luminally expressed MAGI-1 isoforms is preoccupied in vivo by other proteins (18, 22). (ii) The basolateral K+ channels are specifically delivered to the basolateral side by the mechanism that is not clarified yet (12, 23).

The MAGI-1a-long/Kir4.1 interaction was disrupted by phosphorylation. Phosphorylation is thought to take place on the serine 377 in the PDZ-binding motif, and only the PDZ-binding motif with the unphosphorylated serine 377 could interact with the PDZ5 of MAGI-1a-long (Fig. 6B). Either the phosphorylation or the amino acid mutation (to alanine or aspartic acid, which mimics the phosphorylated serine (24, 25)) of the serine 377 disrupted the interaction. In line with our findings, phosphorylation of PDZ-binding motifs in other proteins also changes their interaction with scaffolding proteins and affects their intracellular localization (26, 27).

Interestingly, although the PKA phosphorylation disrupted the MAGI-1a-long/Kir4.1 interaction, it did not change the channel activity of Kir4.1 for a short duration. We previously showed that the PDZ-dependent interaction with scaffolding protein(s) facilitates the localization of the K+ channels on the intracellular compartments just beneath the cell surface but not on the extracellular surface (10, 11). Because of this intracellular localization, the PKA phosphorylation would not change the channel activity for a short duration. The phosphorylation would regulate the process of intracellular localization that affects the channels ready to be expressed on the cell surface. The channels on the compartments just beneath the cell surface could be functional on demand (7).

Serine residues in the CT portion of Kir4.1 other than serine 377, such as serine 343, could be PKA-phosphorylated (28), and might affect the interaction. Less phosphorylation on a S377A-mutated segment under PKA inhibition than PKA stimulation might reflect PKA phosphorylation of these residues (14). However, the difference is too small (p = 0.305) to be considered as the different phosphorylation state of any residues, and serine 377 in the PDZ-binding motif is thought to be the residue that is responsible for the phosphorylation-dependent regulation of the interaction.


Figure 8
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  		FIGURE 8.
Regulation of basolateral K+ channels in distal renal tubules. Phosphorylation-dependent basolateral localization of distal renal tubular K+ channels is schematically summarized. In the distal portion of TAL and the DCT (indicated by green in left panel), MAGI-1a-long (indicated by red dots in right panel) functions as a scaffolding protein for the basolateral K+ channels. In these tubules, K+ channels with unphosphorylated PDZ-binding motifs are anchored on basolateral side. Phosphorylation of serine 377 in PDZ-binding motif impedes the channel anchoring and can diminish K+ reabsorption.

 
In this study, the activity of PKA, which would have changed widely in vitro according to the condition of the cells without extra activation for it, disrupted the MAGI-1a-long/Kir4.1 interaction in HEK293T cells. Therefore, the intracellular signal cascades that affect the activity of PKA in vivo could regulate the activity of the basolateral K+ recycling in distal renal tubules. However, in distal renal tubules, several kinases that can phosphorylate serine residues, such as SGK1, are known to be expressed (29, 30). These kinases might phosphorylate Kir4.1 more efficiently under the conditions that are suitable for their activity. The phosphorylation by these kinases would affect the activity of the basolateral K+ recycling and regulate the renal Na+ reabsorption in vivo in the kidney (29, 30). Supporting this notion, in this study, the animal fed with the diet that could reduce the activity of SGK1 (31) had the increased intrarenal MAGI-1a-long/Kir4.1 interaction.

Although the results of this study suggest the MAGI-1 isoform(s) participate in the localization of the basolateral K+ channel in distal renal tubules and MDCK cells, other scaffolding proteins that contain PDZ domain(s) might participate in the localization. On the basolateral side of MDCK cells and mouse distal renal tubules, the dystrophin-associated protein complex that contains several scaffolding proteins with PDZ domain(s), including {alpha}-syntrophin, is reported to exist (32, 33). Because {alpha}-syntrophin has the PDZ domain that can interact with the PDZ-binding motif of Kir4.1 (34), these proteins might also contribute to the localization of the basolateral K+ channels in distal renal tubules. The contribution of these scaffolding proteins for the localization of the K+ channels remains to be clarified by their direct interaction in distal renal tubules.

In conclusion, MAGI-1a-long functions as a scaffolding protein for the distal renal tubular basolateral K+ channels (Fig. 8). Phosphorylation of the channels impedes the channel scaffolding by MAGI-1a-long and could decrease the basolateral K+ recycling. These findings suggest that the regulatory pathway for the activity of kinases in renal tubules participates in the distal renal tubular Na+/K+ regulation.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY598952 [GenBank] and AY598951.

* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M. T., T. A., and S. I.), by grants from Mishima Kaiun Memorial Foundation (to M. T.), Mochida Memorial Foundation for Medical and Pharmaceutical Research (to M. T.), Japan Heart Foundation/Pfizer Japan Inc. grant for cardiovascular disease research (to M. T.), and Salt Science Research Foundation Grants 0639 and 0734 (to M. T.). 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3. Back

1 To whom correspondence should be addressed: Division of Nephrology, Hypertension, and Endocrinology, Dept. of Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-cho, Aoba-ku, Sendai 980-8574, Japan. Fax: 81-22-717-7168; E-mail: mtanemoto-tky{at}umin.ac.jp.

2 The abbreviations used are: TAL, thick ascending limb; DCT, distal convoluted tubule; PDZ, PSD-95/Dlg/ZO-1; MDCK, Madin-Darby canine kidney; HEK, human embryonic kidney; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; PKA, protein kinase A; RT, reverse transcription; Ni-NTA, nickel-nitrilotriacetic acid; WT, wild type; CT, carboxyl terminus; GFP, green fluorescent protein. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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