标题: PDZ binding motif-dependent localization of K channel on the basolateral side [打印本页] 作者: 轻羽 时间: 2009-4-22 08:08 标题: PDZ binding motif-dependent localization of K channel on the basolateral side
作者：Masayuki Tanemoto, Takaaki Abe,, Tohru Onogawa, and Sadayoshi Ito作者单位：1 Division of Nephrology, Hypertension and Endocrinology, Department of Medicine, and 3 Division of Gastroenterological Surgery, Department of Surgery, Tohoku University Graduate School of Medicine; and 2 PRESTO, Japan Science and Technology Agency 7 v2 r) s% E0 B$ Y6 x 6 o# A$ k/ N+ T; g( W % L" h) |2 L8 L- o: p) a( N+ r
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【摘要】& F T! @, l1 X9 J
Kir5.1, a nonfunctional inwardly rectifying K channel by itself, can form functional channels by assembling with other proteins. We previously showed that Kir5.1 assembled with Kir4.1 and functioned as an acid-base regulator in the kidney. In this study, we examined the intrarenal distribution of Kir5.1 by RT-PCR analysis on dissected nephron segments and immunohistochemical analysis with the specific anti-Kir5.1 antibody. Strong expression of Kir5.1 was detected in distal convoluted tubules, and weak expression was also detected in thick ascending limb of Henle's loop. Colocalization of Kir5.1 with Kir4.1 indicated expression of Kir5.1/Kir4.1 heteromer in these nephron segments. In a renal epithelial cell line, Madin-Darby canine kidney cells, heteromer formation with Kir4.1 changed the localization of Kir5.1 from intracellular components to the cell surface. The COOH-terminal cytoplasmic portion that includes the PDZ binding motif of Kir4.1 was responsible for this intracellular localization. These data suggest the signals on the COOH terminus of Kir4.1, including PDZ binding motif, determine the intracellular localization of Kir5.1/Kir4.1 heteromer in distal tubules.6 W" G1 Z2 |/ s6 I
) a8 I! z! L. M4 m6 q7 T4 m" a7 q 0 ^1 a6 n" Y# g& O6 T+ w ( w- {' A8 y4 P& q' U4 ^" P3 Q" {. YKir5.1/Kir4.1 heteromer; renal distal tubules; intracellular localization; PDZ domain . Z% e/ \# L8 C$ I2 w 【关键词】 motifdependent localization basolateral+ @: H/ Q( g" i* x- ] @
THE INWARDLY RECTIFYING K channel subunit family (Kir) can be classified into four major groups: K transporters (Kir1.0/4.0 subfamily), background K channels (Kir2.0 subfamily), G protein-gated K channels (Kir3.0 subfamily), and ATP-sensitive K channels (Kir6.0 subfamily) ( 9 ). K transporter members Kir1.1 and Kir4.1 are expressed in renal distal tubules and function as key molecules for renal ion transport ( 8, 25 ). The functional loss of K transporters induces imbalance of not only potassium but also other ions such as sodium and proton, and mutation in the gene coding Kir1.1 causes Bartter's syndrome, which is characterized by hypokalemic alkalosis ( 7, 18 ). Kir5.1, which does not belong to these major groups and does not form a functional K channel by itself, can form a functional channel through assembly with other proteins; e.g., heteromeric assembly with K transporters Kir4.1 and Kir4.2 ( 1, 14, 15, 20, 23, 24 ), and assembly of homomeric Kir5.1 with a member of anchoring protein family, PSD-95 ( 19 ). Intracellular modification changes functional activity of these channels and may take part in physiological regulation of ion transport. Acidification within the physiological range reduced the channel activity of Kir5.1/Kir4.0 heteromer ( 1, 14, 15, 20, 23, 24 ), and protein kinase A-mediated phosphorylation of Kir5.1 inhibited the channel activity of Kir5.1/PSD-95 complex ( 19 ). 6 _* z/ N! K4 j3 ^; @+ ] $ K* Q5 u1 I9 Z9 n* Z9 ~In the previous report, we raised an anti-Kir5.1-specific antibody and showed that Kir5.1 could form a heteromer with a member of K transporters, Kir4.1, and function as an intracellular pH-dependent ion transporter in the kidney ( 20 ). Using this antibody, we also showed that Kir5.1 assembled with PSD-95 in the brain ( 19 ). However, the antibody we generated previously was not suitable for immunohistochemical analysis, and the precise intrarenal localization of Kir5.1 has not been clarified. Because other members of K transporters and several members of the anchoring protein family have been shown to be expressed in the kidney ( 17, 22, 26 ), it is possible that Kir5.1 forms functional K channels in the kidney with the aid of these proteins ( 3, 14 ). To evaluate this possibility, we examined the intrarenal expression of Kir5.1 mRNA by RT-PCR on dissected nephron segments and also at the protein level by immunohistochemical analysis by raising a new anti-Kir5.1-specific antibody. Furthermore, we evaluated the mechanism of intracellular localization of Kir5.1 in renal tubules, using Madin-Darby canine kidney (MDCK) cells as a model of renal epithelia. , j8 V7 r- w; U+ O* j , \7 v1 N5 E6 b6 H/ VMATERIALS AND METHODS 9 X4 _; @. ?8 E* }, ~. e: N" X8 ~6 z
RT-PCR analysis. Microdissection of individual nephron segments and reverse transcription (RT) were performed as described previously ( 21 ). The following nephron segments were microdissected: glomerulus (Gl), proximal convoluted tubules (PCT), proximal straight tubules (PST), medullary and cortical thick ascending limb of Henle's loop (MTAL, CTAL), distal convoluted tubules (DCT), and cortical, outer medullary, and inner medullary collecting ducts (CCD, OMCD, IMCD). Total RNA was extracted from cells by TRIzol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed (RT) with oligo(dT) 12-18 primer by Superscript II RT (Invitrogen) at 42°C for 60 min. PCR was performed using r Taq (Takara Bio, Shiga, Japan). Detection of Kir5.1 expression was performed with primer pairs of a sense primer (5'-ATACTGGTGACTCTACTGGG-3') and an antisense primer (5'-TCTCCTCCTGGTGTTGGTG-3') on the following schedule: denatured at 95°C for 2 min and amplified by 50 cycles of 94°C for 15 s, 58°C for 30 s, and 72°C for 45 s. Detection of PSD-93 expression was performed with primer pairs of a sense primer (5'-AATATGAATTTGAAGAAATTAC-3') and an antisense primer (5'-TAAGGATCAGTCATATAAAT-3') on the following schedule: denatured at 95°C for 2 min and amplified by 35 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 90 s. PCR reactions were also performed using a pair of -actin primers as a control to adjust the uniformity of amplification. The number of templates for dissected tubules was adjusted according to the amount of -actin cDNA amplified from each sample. The primers of -actin span one intron to discriminate genomic DNA. The PCR products were analyzed by agarose gels and visualized by staining with ethidium bromide.( c7 H R8 g* m* ]
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Gultathione S-transferase pull-down analysis. Gultathione S -transferase (GST) fusion proteins of PDZ proteins were constructed by subcloning PCR-amplified DNA fragments into pGEX-5 3 vector (Amersham Biosciences, Piscataway, NJ). The region from the first PDZ domain to the COOH terminus of PSD-93 or PSD-95 was fused to GST. Pull-down analysis was performed as previously described ( 19 ). . n0 t8 _; J }: E& g, c1 R E; Y! U( L, h
Antibodies. Polyclonal anti-Kir5.1 antibody was raised in rabbits against the synthetic peptide NVDSKYPGYPPEHAIAEKR that corresponds to the 12-30th amino acids of Kir5.1 and affinity-purified as reported previously ( 20 ). Polyclonal anti-Kir4.1 antibody was purchased from Sigma (St. Louis, MO). Polyclonal anti-NKCC2 and NCC antibodies were purchased from Chemicon International (Temecula, CA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was purchased from Amersham Biosciences. Alexa Fluor 594 anti-rabbit IgG (Fab') 2 and fluoresein isothiocyanate (FITC)-labeled anti-rabbit IgG were purchased from Molecular Probes (Eugene, OR) and DAKO (Glostrup, Denmark), respectively.4 i( Y# ~5 a& E3 o7 `
0 z: X( _7 Y' STransient expression of Kirs in HEK293T cells and MDCK cells. Coding regions of wild-type Kir4.1 and wild-type Kir5.1 were amplified by RT-PCR from rat kidney mRNA and deletion-mutants of Kir4.1 (3 or 105 amino acids are deleted from the COOH terminus) were also PCR amplified. These PCR products were subcloned into mammalian cell expression vectors pCDNA3 (Invitrogen) and pEGFP-C1 (Clontech Laboratories, Palo Alto, CA) as described previously ( 19 ). These plasmid vectors were transfected to HEK293T cells or MDCK cells by using LipofectAMINE 2000 (Invitrogen). For cotransfection of Kir4.1 with Kir5.1, five times more Kir4.1 cDNA was cotransfected with Kir5.1 cDNA ( 20 ). Membrane preparation and microscopic observation were usually conducted 2-3 days after transfection.+ F, ]1 z& o/ q' C" G
/ l+ K' G3 e9 r# n" g2 PImmunoblot analysis. An adult male Sprague-Dawley rat was anesthetized by ether and killed by decapitation, according to the regulations of the Animal Care Committee of Tohoku University Medical School. Membrane preparations of Kir4.1- or Kir5.1-transfected HEK293T cells and whole rat kidney were obtained as described previously ( 20 ). The membrane proteins were solubilized in a lysis buffer containing 150 mM NaCl, 50 mM Tris·HCl (pH 7.4), 1 µg/ml aprotinin, 100 µg/ml PMSF, 0.02% sodium azide, 0.1% SDS, 0.5% sodium deoxycholate, and 1% Triton X-100. About 10 and 40 µg of solubilized membrane proteins from HEK293T and rat kidney, respectively, were analyzed by Western blotting. The blot was incubated for 12 h at 4°C with a blocking buffer [80 mM NaCl, 50 mM Tris·HCl (pH 7.5), 5% (wt/vol) skim milk, and 0.2% Triton X-100] and then with the buffer containing the primary antibody (0.5 µg/ml) for 12 h at 4°C. The blot was then incubated with the HRP-conjugated anti-rabbit IgG (1:5,000 dilution) for 1 h at room temperature. Enhanced chemiluminescence kit was used for detection (Amersham Biosciences).- K/ K" e- ^+ F7 m; a
- h5 T3 h& R% T, V% ~: IImmunohistochemical analysis. An adult Sprague-Dawley rat was anesthetized according to the regulations of the Animal Care Committee of Tohoku University Medical School and then perfused transcardially with 0.9% saline and followed by 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.4). The specimens were immersed in 30% sucrose/0.1 M phosphate buffer. The sections were made at a thickness of 10 µm with a cryostat and mounted onto gelatin-coated slide glasses. After incubation in PBS containing 0.05% Triton X-100 (PBST), the sections were then exposed to 5% normal goat serum for 30 min to block nonspecific staining. The sections were incubated with the first antibody at a concentration of 1 (for anti-Kir4.1) or 50 (for anti-NKCC2 and anti-NCC) µg/ml followed by incubation with an excess (1:20 dilution) of Alexa Fluor 594 anti-rabbit IgG (Fab') 2 to saturate the epitopes of the antibodies. After being washed extensively with PBS, the sections were subsequently incubated with anti-Kir5.1 at a final concentration of 1 µg/ml, followed by incubation with FITC-labeled anti-rabbit IgG at a 1:100 dilution in 5% bovine serum albumin/PBST. The sections were washed three times with PBS and observed by confocal microscopy. The saturation of the precedent antibody was confirmed by preliminary experiments that did not detect any labeling with FITC by subsequent incubation with control rabbit IgG. + m7 o1 V" r: `" } & w N+ s9 M+ K! \, C J. S/ zConfocal microscopic observation. MDCK cells were plated on polycarbonate Millicell transwell filters (Millipore, Bedford, MA), and after 12 h incubation plasmid vectors were transfected. After the confluent growth was confirmed, MDCK cells were rinsed with PBS and fixed in 4% paraformaldehide (pH 7.4 with sodium phosphate). The sample preparation for confocal microscopic observation was performed as previously described ( 19 ). Confocal microscopic observation of immunohistochemical sections and MDCK cells was performed on a model LSM 5 PASCAL (Carl Zeiss, Jena, Germany). . X# Z9 G$ Y0 u4 d. s $ s, w a/ g* Y% ZRESULTS7 t4 N9 R; V& }% {
* c/ j* q% _( m' h3 K8 `8 {% Z; ~2 eExpression of Kir5.1 mRNA in the kidney. To clarify the mRNA expression of Kir5.1 in the kidney, we performed RT-PCR analysis on dissected nephron segments as described in MATERIALS AND METHODS. The samples of Gl, however, may have contained a distal end of CTAL and the beginning of DCT, because CTAL returned to its parental Gl and attached to the extraglomerular mesangium. More than three samples of each nephron segment were PCR analyzed with specific primer pairs of Kir5.1. The result of the amplification for each segment was semiquantitatively adjusted according to the amount of -actin and summarized in Fig. 1. The samples that had not been reverse transcribed before PCR were analyzed as negative controls, and the plasmid containing cDNA of Kir5.1 was PCR-amplified as positive control. An expression of Kir5.1 mRNA was detected in the samples of Gl, DCT, and CCD ( lanes 1, 6, and 7 in Fig. 1, top, respectively), and a weak expression was also detected in the samples of CTAL ( lane 5, top ). Amplification by 15 more cycles of PCR detected an expression of Kir5.1 mRNA in the samples of MTAL but not PCT, PST, OMCD, and CCD ( Fig. 1, bottom ). . E% \* x) @/ X. [% R & s/ }$ [: @ o+ u) E0 OFig. 1. Expression of Kir5.1 mRNA in nephron segments. Top and bottom : result of RT-PCR amplification from dissected nephron segments using specific primer pairs for Kir5.1. The products were analyzed by electrophoresis and visualized by ethidium bromide staining. The amplified products of Kir5.1 were detected in glomerulus (Gl), cortical thick ascending limb (CTAL), distal convoluted tubules (DCT), and cortical collecting duct (CCD) at the same size of positive control. Expression was also detected in medullary TAL (MTAL) but not in proximal convoluted tubules (PCT), proximal straight tubules (PST), outer and inner medullary collecting ducts (OMCD and IMCD) by 15 cycles more PCR amplification. Each lane represents nephron segment of 1 : Gl, 2 : PCT, 3 : PST, 4 : MTAL, 5 : CTAL, 6 : DCT, 7 : CCD, 8 : OMCD, 9 : IMCD, 10 : RT (-) samples as negative control, and 11 : cDNA of Kir5.1 as positive control.& G1 B9 A& j" |# h" X$ O
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Characterization of anti-Kir5.1 antibody. To examine the distribution of Kir5.1 more precisely at a protein level, we raised a polyclonal antibody against a synthetic peptide corresponding to a part of the NH 2 terminus of Kir5.1. After affinity purification, we characterized this antibody by immunoblotting ( Fig. 2 A ). The antibody (anti-Kir5.1 antibody) recognized a single band in the lysate of HEK293T cells transfected with Kir5.1. The size of this band was around 49 kDa, which is the predicted size of Kir5.1 ( lane 2 in Fig. 2, top ). The band of the same size was also detected in the lysate from the membrane fraction of rat kidney ( lane 3 in Fig. 2, top ). No bands were detected in the lysate from HEK293T cells transfected with Kir4.1 ( lane 1 in Fig. 2, top ). Preincubation with the excess amount of antigenic peptide completely blocked the immunoreactivity of the anti-Kir5.1 antibody with these bands ( Fig. 2, bottom ). The specificity of the anti-Kir5.1 antibody was further analyzed with immunocytochemistry ( Fig. 2 B ). Kir5.1 expression in HEK293T was detected diffusely in the cytoplasm with the anti-Kir5.1 antibody ( Fig. 2 B, left ), but no immunoreactivity was detected in the cells transfected with control plasmid vector ( Fig. 2 B, right ).$ C3 w8 j( I w$ e" c, _) ~1 P
9 f9 N9 J. T }0 T& |1 PFig. 2. Specific recognition of Kir5.1 by anti-Kir5.1 antibody. A : specific single band about 49 kDa was detected in samples of Kir5.1-transfected HEK293T cells ( lane 2 ) and rat kidney ( lane 3 ) but not Kir4.1-transfected HEK293T cells ( lane 1 ) by Western blot analysis using the anti-Kir5.1 antibody ( top ). These detections were specifically inhibited by preincubation of the antibody with specific antigenic peptide ( bottom ). B : anti-Kir5.1 antibody specifically detected immunoreactivity diffusely in the cytoplasm of Kir5.1-transfected but not mock-transfected HEK293T cells. Bottom : brightfield view of corresponding top. Scale bar indicates 10 µm. ?3 }0 w2 z) ]* F% Y
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Intrarenal localization of Kir5.1. Using this anti-Kir5.1-specific antibody, we examined the colocalization of Kir5.1 with Kir4.1 in rat kidney with an immunofluorescent method ( Fig. 3 ). A positive immunoreactivity of Kir5.1 was observed in cortex and outer medulla, but not in inner medulla ( Fig. 3 A, left ). The immunoreactivity in the medullary region was faint, and the intensity of immunofluorescence increased as tubules went into the cortical region (white arrowhead). The strongest immunoreactivity was detected in the tubules near the vascular pole of Gl, but no apparent immunoreactivity was observed in the intraglomerular area. As shown at higher magnifications, the immunoreactivity of Kir5.1 was observed near the basolateral side in these segments ( Fig. 3 B ). Each epithelium in these renal tubules showed broad rather than thin linear immunoreactivity of Kir5.1 near the basolateral side, but no apparent immunoreactivity was detected on the luminal side. This diffuse immunoreactivity of Kir5.1 near the basal side is thought to reflect the tangled invagination of basolateral membrane in this nephron segment. A positive immunoreactivity of Kir4.1 was also observed in the same nephron segments ( Fig. 3, A and B, middle ) and colocalized with Kir5.1 in these nephron segments as prominently shown by yellow signal in the merged view ( Fig. 3, A and B, right ). $ o+ b2 y0 d6 @( W# e- L & o+ Z$ G3 ^0 S* I% RFig. 3. Intrarenal localization of Kir5.1 in rat kidney. A : localization of Kir5.1 in rat kidney was observed by using the anti-Kir5.1-specific antibody. The strong immunoreactivity of Kir5.1 was detected in the cortex. Weak immunoreactivity was also observed in outer medulla, and the immunoreactivity increased as the tubules went into cortex (arrowhead). Scale bar: 100 µm. B : higher magnification of cortex and outer-inner boundary indicates localization of Kir5.1 near the basal side of the tubules at the vascular pole of glomerulus. The immunoreactivity was detected not linearly on the basal membrane but diffusely near the basal side of epithelia, which is postulated to reflect the tangled invagination of basolateral membrane in this nephron segment. G, glomerulus; OM, outer medulla; IM, inner medulla. Scale bar: 100 µm. C : precise localization of Kir5.1 in distal tubules was shown using anti-NKCC2 or NCC antibody as a marker. The immunoreactivity of Kir5.1 was detected diffusely near the basal side of distal tubules from CTAL including macula densa cells (white arrowhead), where NKCC2 is expressed on the apical side ( top ), to DCT, where NCC is expressed on the apical side ( bottom ). Scale bar: 20 µm. ) J- i% C9 g: S5 F9 R8 T' Z. }1 S1 N! i- k, t
The immunoreactivity of Kir5.1 was detected in the nephron segments that express NKCC2 or NCC on the luminal side of epithelia ( Fig. 3 C ). Kir5.1 did not colocalize with these luminal transporters in TAL and DCT, as shown in the merged views. These results confirmed expression of Kir5.1 on the basolateral side of distal tubules from MTAL to DCT. Especially, the immunoreactivity of Kir5.1 was detected in macula densa (MD) at the vascular pole of the glomelurus (white arrowhead in Fig. 3 C, top ).0 z" u+ C# G. H7 D3 ]8 ^. D. p7 d
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Kir4.1-induced clustered distribution of Kir5.1 in MDCK cells. The underlying mechanism for intracellular localization of Kir5.1 channel was further examined by an in vitro expression system. We used MDCK cell as a model of renal epithelium, because it is a mammalian cell-line derived from renal epithelium. Kir5.1 to which green fluorescent protein (GFP) was tagged at the NH 2 terminus (G-Kir5.1) was transiently expressed in MDCK cells, and its intracellular distribution was observed after the confluent growth and sheet formation of cells were established ( Fig. 4 A ). When G-Kir5.1 was expressed alone, it was localized diffusely in the cytoplasm, while GFP tagged Kir4.1 (G-Kir4.1) showed dominant clustered distribution on cell surface. When G-Kir5.1 was cotransfected with Kir4.1, clustered distribution of G-Kir5.1 could be observed on cell surface in about 10% of cells that expressed G-Kir5.1 (G-Kir5.1 Kir4.1). This indicates that heteromer formation with Kir4.1 can locate G-Kir5.1/Kir4.1 heteromer on the intracellular transport system to the cell surface. 6 c: y8 P! \+ ]9 R/ m1 P, ~/ ^! J; N f# O/ _1 v- v! V8 l
Fig. 4. Heteromer formation with Kir4.1 induces clustered distribution of Kir5.1 in Madin-Darby canine kidney (MDCK) cells. A : top and bottom show top and side views of cells. The Kir subunits expressed in MDCK cells are indicated on the top. When expressed alone GFP-tagged Kir5.1 distributed diffusely in the cytoplasm (G-Kir5.1), whereas GFP-tagged Kir4.1 showed clustered localization around the nucleus and cell surface (G-Kir4.1). When cotransfected with Kir4.1 (G-Kir5.1 Kir4.1), clustered distribution of Kir5.1 around the nucleus and cell surface (arrowheads) could be observed. Deletion of COOH-terminal PDZ binding motif changed the localization and peri-nuclear clustered distribution was observed (G-Kir4.1 CT3). The clustered distribution of Kir4.1 was diminished when a COOH-terminal 105-amino acid of Kir4.1 was deleted (G-Kir4.1 CT105). Scale bars: 10 µm. B : RT-PCR analysis revealed the expression of PSD-93 in MDCK cells ( left ). At the same size of PCR products from rat brain as a positive control ( lane 1 ), PCR products were obtained from MDCK cells ( lane 2 ). Lane 3 : samples without RT as a negative control. GST pull-down assay shows no apparent interaction between Kir4.1 and PSD-93 ( right ). GST tagged PSD-95 can ( lane 3 ) but GST itself ( lane 1 ) and GST-tagged PSD-93 ( lane 2 ) cannot bind to GFP-tagged Kir4.1. : E0 e) @: g8 d0 f; M7 M8 a$ e5 |/ g0 p4 d
The above patterns of the intracellular distribution of Kir channels in MDCK cells suggest that some signals of Kir4.1 determine intracellular localization of these channels. To identify these signals, intracellular distribution of the COOH-terminal (CT) deletion mutants of Kir4.1 was examined ( Fig. 4 A ). Only the deletion of a PDZ domain (PSD-95/Dlg/ZO-1 also known as Disc-Large Homology Repeats or GLGF repeats)-binding motif (SNV) (G-Kir4.1 CT3) changed the intracellular distribution of Kir4.1, and the mutant channel showed perinuclear clustered distribution. This change of intracellular localization by deletion of the PDZ binding motif indicated that some anchoring proteins containing PDZ domain(s) determine cell-surface localization of Kir channels in renal epithelia. Interestingly, further deletion of 102 amino acids from the COOH terminus diminished the perinuclear clustered distribution and induced diffuse cytoplasmic distribution (G-Kir4.1 CT105).1 |: O, P% U: w
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Because PSD-93, a member of PDZ domain-containing anchoring proteins, was reported to be expressed on the basolaterel side of renal tubules including CTAL and DCT, we examined the possible role of PSD-93 for the cell-surface localization of Kir4.1 in MDCK cells ( Fig. 4 B ). Although expression of PSD-93 in MDCK cells was detected by RT-PCR analysis ( Fig. 4 B, left ), GST pull-down analysis revealed no direct interaction between Kir4.1 and PSD-93 ( Fig. 4 B, right ).8 e+ T0 p8 \6 N% o0 U; y/ j4 z
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DISCUSSION 4 O, H8 ^; P, E% R/ A l 1 d' [# p5 ^' |, m) U u( GThe intrarenal distribution of Kir5.1 according to the results of the present study is summarized in Fig. 5. Although we could not detect Kir5.1 mRNA expression in renal tubules of outer medulla in lower amplification of PCR analysis, we could detect the expression by additional amplification in MTAL but not PST and OMCD. Considering this result of RT-PCR analysis, the corresponding nephron segments that showed faint positive immunoreactivity of Kir5.1 in the outer medullary area would be MTAL that begins from the outer-inner boundary. As indicated by the immunoreactivity and the semiquantitative PCR analysis, the amount of expression increased as the tubule approached its parent Gl. The strongest expression was detected near the vascular pole of Gl in the final portion of CTAL, which includes MD, and DCT. ) X, J2 |7 I/ ] u% ]/ n; W+ ^0 {7 U4 `* Y4 c% d, d7 C
Fig. 5. Summary of intrarenal expression of Kir5.1. Intrarenal localization of Kir5.1 was shown schematically. Kir5.1 was expressed mainly on the basolaterel side of DCT and the early segment of CCD. Weak expression of Kir5.1 was also detected from MTAL to CTAL. Kir5.1 colocalizes with Kir4.1 and is postulated to exist as a Kir5.1/Kir4.1 heteromer in these renal tubules. : _3 a, ?. A3 Q( h( P! E8 D . u7 ]$ v. g2 KAs the RT-PCR analysis detected expression of Kir5.1 in CCD, Kir5.1 may be expressed in the early segment of CCD where we did not describe the distribution of Kir5.1 on the immunohistochemical analysis. The immunoreactivity of Kir5.1 would be detected in the early segment of CCD on the immunohistochemical analysis, because the epithelium from DCT to CCD is known to be continuous and the cells that show typical characters of DCT are also located in the early segment of CCD ( 10 ). We could not describe this localization of Kir5.1 in the early segment of CCD, because the epithelium in the early segment of CCD is difficult to distinguish from DCT in thin slice sections. % _8 g* Q$ ]# \3 |* Y: o/ D9 p$ w! x# g' y! V( p, `+ ~" G
Although the RT-PCR analysis detected the expression of Kir5.1 in the microdissected samples of Gl, no apparent immunoreactivity of Kir5.1 was detected in the intraglomerular area. We think the detection of Kir5.1 expression in RT-PCR analysis reflects the contamination of the distal end of CTAL and the beginning of DCT. Structurally, the distal tubule returns to its parent Gl and is attached to the extraglomerular mesangium at the vascular pole ( 10 ). It is technically difficult to completely separate this tubular segment, especially MD at the end of CTAL, from Gl. Therefore, the dissected samples of Gl contained a part of CTAL and DCT including MD that is attached to the extramesangial matrix at the vascular pole of Gl. Even small pieces of contamination of attached tubular segments would be enough to be moderately detected from microdissected Gl samples in the RT-PCR analysis. The result of immunohistochemistry that shows strong immunoreactivity of Kir5.1 in MD supports this notion. - Y* _- h7 ] t$ {$ q) X C p- O% P, {. ~* t
Previous studies on intrarenal localization of Kir4.1 have shown its immunoreactivity on the basolateral side of epithelia from DCT to the early segment of CCD ( 8 ). We could also detect the immunoreactivity of Kir4.1 in these nephron segments using the antibody that was raised against the different part of Kir4.1 from the antibody used in the previous report. Using this antibody, we could also detect faint immunoreactivity in other nephron segments that are deduced to be MTAL and CTAL. This discrepancy probably reflects weak immunoreactivity of Kir4.1 in TALs. The immunoreactivity of Kir4.1 might be too weak to be discriminated by the enzymatic detection method used in the previous report.% B9 {' I7 A) J C
1 e: _/ a* P5 \* |The present study demonstrates that Kir5.1 colocalizes with Kir4.1 in all these distal tubules including TALs. Because we already showed the interaction of Kir5.1 with Kir4.1 in the kidney at the protein level using the immunoprecipitation method ( 20 ), the result of the present study indicates expression of a Kir5.1/Kir4.1 heteromer near the basolateral side of these distal tubules. We recently reported the channel properties of native K channels expressed on the basolateral membrane of DCT ( 11 ). Most channels showed properties that resemble those of a Kir5.1/Kir4.1 heteromer but not a Kir4.1 homomer; e.g., 1 ) a large single-channel conductance, 2 ) short open times and long closed times, and 3 ) a high sensitivity to intracellular pH. The similarity of electrophysiological characters of basolateral K channels of DCT to a Kir5.1/Kir4.1 heteromer supports the notion that a Kir5.1/Kir4.1 heteromer is expressed on the basolateral membrane of distal tubules. ! \% U S$ v: ~- K. O# L+ d - R& f: B6 ], v: p6 z1 E" Y! @TALs and DCT are the nephron segments where sodium reabsorption takes place. In these nephron segments, Na -K -ATPase drives sodium reabsorption and Kir channels are thought to assist sodium reabsorption by excluding potassium ions that enter into tubular cells in the process of sodium reabsorption. The channel activity of Kir channels would therefore affect the sodium transport in these nephron segments. Because the channel activity of the Kir5.1/Kir4.1 heteromer is regulated by intracellular pH within the physiological range ( 20, 23, 24 ), systemic acid-base derangement would affect sodium reabsorption in these nephron segments. As seen in the case of loop and thiazide diuretic usage, which inhibit sodium reabsorption from TALs and DCT, respectively, increased delivery of sodium to collecting ducts induces proton excretion into urine. Therefore, from the teleological point of view, the expression of a Kir5.1/Kir4.1 heteromer in TALs and DCT is appropriate for the kidney to regulate an acid-base balance. In the conditions of acidemia, sodium absorption was reduced by inhibition of Kir channel activity. The consequent increase in sodium delivery to the collecting ducts contributes to exclusion of excess protons. The reverse process would take place under the conditions of alkalemia. In this way, a Kir5.1/Kir4.1 heteromer may be involved in renal compensatory mechanisms to maintain the systemic acid-base homeostasis.# J5 O+ ]6 X" \4 u8 {# [* Z
9 X5 M9 m$ y$ L/ c m) Z2 Z6 jPreviously, RT-PCR analysis on dissected nephron segments of human kidney detected Kir5.1 mRNA expression in PCT ( 5 ). In addition, using an anti-Kir5.1 antibody raised against a different part of Kir5.1 from ours, positive immunostaining of Kir5.1 had been reported in rat PCT ( 23 ). In the present experiment, we could not detect Kir5.1 expression in PCT of rat kidney by both RT-PCR and immunostaining analyses. We cannot explain these discrepancies precisely. However, these may be attributable to differences in the species used, the sample preparations, the primers used for RT-PCR, and the quality of antibodies. 4 a T. k% f g. i* X ' E4 \8 N: R( W! q4 p5 pUsing MDCK cells as a model of renal epithelia, we analyzed the underlying mechanism to form a functional Kir5.1/Kir4.1 heteromer. As in the case of PSD-95, which induced cell-surface expression of Kir5.1 in HEK293T cells ( 19 ), Kir4.1 also changed intracellular localization of Kir5.1. Kir4.1 induced dominant clustered distribution of Kir5.1 in MDCK cells. Only the deletion of three COOH-terminal amino acids (Kir4.1 CT3), which correspond to the PDZ-binding motif, changed the distribution of the Kir channel and the deletion mutant clustered predominantly around the nucleus. Further deletion of 102 amino acids from the cytoplasmic COOH terminus (Kir4.1 CT105) diminished clustered distribution of the channel protein. These results show that heteromer formation with Kir4.1 is crucial for intracellular localization of a Kir5.1/Kir4.1 heteromer and that the cytoplasmic COOH-terminal domain of Kir4.1 played an indispensable role in this localization.1 Y/ S8 L! D- Z4 X+ I
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The intracellular distribution of mutant channels in MDCK cells may indicate that Kir4.1 but not Kir5.1 contains several signals for intracellular transport to the cell surface. Proteins that are destined for cell-surface expression are transported through the intracellular transport system including the Golgi apparatus. Because the Golgi apparatus locates around the nucleus, the perinuclear clustered localization of Kir4.1 CT3 may indicate that three COOH-terminal amino acids (SNV) are necessary for cell-surface expression but not for the transport to the Golgi apparatus. Because the COOH-terminal SNV is a PDZ domain-binding motif ( 4, 16 ), some member(s) of anchoring proteins containing these domain(s) are candidates that determine the cell-surface localization of Kir channels in MDCK cells and renal distal tubules. Although the expression of PSD-93, a member of the PDZ domain-containing anchoring proteins that was reported to be expressed on the basolaterel side of renal distal tubules ( 22 ), was detected in MDCK cells, there was no direct interaction between Kir4.1 and PSD-93. Therefore, some other member(s) of the PDZ domain-containing anchoring proteins may be involved in basolateral localization of Kir channels in renal distal tubules and MDCK cells. ' L; ^; L+ W7 [$ E) M, f: I, B7 K9 M! j: U! K" Y, ^7 g: `9 M
The diffuse distribution of Kir4.1 CT105 in the cytoplasm indicates that some signal(s) in the COOH-terminal 102 amino acids (from -4 to -105 from the COOH end) is responsible for intracellular transport of Kir4.1 from ribosome to Golgi apparatus. Several different intracellular localization signals of membrane proteins have already been reported ( 2, 12 ). Many of these signals are characteristic arrays of amino acid sequence that exist on the cytoplasmic domains of membrane proteins. Among these signals, the tyrosine-based motif and di-leucine motifs were recognized to function as intracellular transport signals for basolateral targeting ( 2, 6, 13 ). In COOH-terminal 102 amino acids (-4 to -105), Kir4.1 contains several sequences that resemble these motifs. We could not clarify which of these motifs determined intracellular transport of a Kir5.1/Kir4.1 heteromer. Several motifs among them may cooperatively take part in intracellular transport ( 2, 12 ). Both of the basolateral targeting and the anchoring signals should be necessary for the efficient basolateral localization of a Kir5.1/Kir4.1 heteromer in the epithelium of distal tubules.$ x* g4 K+ s0 p3 C5 P
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In conclusion, Kir5.1 was expressed near the basolateral side of distal tubules from MTAL to the early segment of CCD. In these distal tubules, Kir5.1 is postulated to exist as a Kir5.1/Kir4.1 heteromer, an intracellular pH-regulated K channel, and the cytoplasmic COOH-terminal portion of Kir4.1 presumably determines the intracellular localization of this heteromer. % S2 m- n B2 k" b; d5 _! ]$ j( W% t+ d% a
GRANTS 1 ~9 I2 M* G5 A7 ]0 s8 a" e ! x' `2 ] P, X( AThis work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (no. 15689005 to M. Tanemoto, no. 14370777 to T. Abe, and no. 15390264 to S. Ito) and the Yamanouchi Foundation for Research on Metabolic Disorders (M. Tanemoto).* R/ X; E" {+ O- }0 `
【参考文献】- c) k# v3 [8 T1 n% M' P, q" Y
Bond CT, Pessia M, Xia XM, Lagrutta A, Kavanaugh MP, and Adelman JP. Cloning and expression of a family of inward rectifier potassium channels. Receptors Channels 2: 183-191, 1994. / Z8 ^" q, F' }- ~: N# y" F2 O # h+ H: X8 O3 k: j* n" X4 }* G6 s7 m+ p! @* ` J
7 B0 X$ l) \$ e+ U
Brown D and Breton S. Sorting proteins to their target membranes. Kidney Int 57: 816-824, 2000., M* i+ n. c+ M" D/ Z l4 c
1 v0 s: b9 H" `& ]- P/ s! `0 i 8 z* U& W _% z7 i$ H( E# y# Y : P5 |9 O) }+ k; k4 iConnors NC and Kofuji P. Dystrophin Dp71 is critical for the clustered localization of potassium channels in retinal glial cells. J Neurosci 22: 4321-4327, 2002. : {3 j7 o# r5 t, o$ S- j! p 2 N% V" J3 F) m0 |4 Q% a7 i" n" t3 `& B
( N5 A# v/ q3 o) }* B! P Y9 rCraven SE and Bredt DS. PDZ proteins organize synaptic signaling pathways. Cell 93: 495-498, 1998.- F! }& Z/ Z6 y0 ~ @+ l1 {
0 K& h/ v1 d7 f8 @# [) m R$ D9 [
$ z/ n5 M( s7 ~# g5 x
`. c2 Y3 g; ^# {
Derst C, Karschin C, Wischmeyer E, Hirsch JR, Preisig-Muller R, Rajan S, Engel H, Grzeschik K, Daut J, and Karschin A. Genetic and functional linkage of Kir5.1 and Kir21 channel subunits. FEBS Lett 491: 305-311, 2001.: _: P; _0 K9 i# E. ~
% W! ], T4 P. R4 w- P! z
" s3 [4 }& @/ Q- u, x: P# I$ H7 M. u* R( T$ i- @* s
Hunziker W and Fumey C. A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc receptors in MDCK cells. EMBO J 13: 2963-2967, 1994.4 o+ H0 f) m; S9 o$ n! S: j/ p
& P/ R, n9 x% w$ H/ ?3 _! L% M
5 J& r% `; W: i7 S 6 ~# B. ~- {' R) ^7 Q+ ]International Collaborative Study Group for Bartter-like Syndromes. Mutations in the gene encoding the inwardly-rectifying renal potassium channel ROMK cause the antenatal variant of Bartter syndrome: evidence for genetic heterogeneity. Hum Mol Genet 6: 17-26, 1997. 9 i1 m% K6 o. q! k6 |) X$ o3 I7 b( t5 X. R& R3 E* z* d1 r8 i
C/ p. Z9 a0 K9 Z( f$ |0 c. L! v- E4 _9 w0 K: p" A' O& j
Ito M, Inanobe A, Horio Y, Hibino H, Isomoto S, Ito H, Mori K, Tonosaki A, Tomoike H, and Kurachi Y. Immunolocalization of an inwardly rectifying K channel, K(AB)-2 (Kir4.1), in the basolateral membrane of renal distal tubular epithelia. FEBS Lett 388: 11-15, 1996.- h5 f" K; M0 k$ O
* j' ~4 U2 z9 A* d q- O4 `
2 R" i! K+ G7 T, @ ' w. @/ i( {- C+ J6 HJan LY and Jan YN. Cloned potassium channels from eukaryotes and prokaryotes. Annu Rev Neurosci 20: 91-123, 1997., U* a7 B" x* I, }: W
/ q4 f. ?0 M" W* ?9 l, Q( G
' r7 Z, r4 z; N
* V/ ~4 D6 G: _. W% X4 S, xKriz W and Kaissling B. Structural organization of the mammalian kidney. In: The Kidney (3rd ed.), edited by Seldin DW and Giebisch G. Philadelphia: Lippincott Williams & Wilkins, 2000, p. 587-654.9 x9 E5 L. j6 H; v: Q
, O& i0 B9 h, W% @& f- U5 W( N+ `$ L, u; v/ `
9 ]) J" u7 S1 @1 `
Lourdel S, Paulais M, Cluzeaud F, Bens M, Tanemoto M, Kurachi Y, Vandewalle A, and Teulon J. An inward rectifier K channel at the basolateral membrane of the mouse distal convoluted tubule: similarities with Kir4-Kir5.1 heteromeric channels. J Physiol 538: 391-404, 2002. 2 V5 D; z( p4 |6 b) i) m1 \7 c4 f, I0 _, d$ o
* b% \: S9 r/ L; K 9 N/ j l5 \0 Y' {9 L0 r3 X% [Matter K. Epithelial polarity: sorting out the sorters. Curr Biol 10: R39-R42, 2000.5 {, [/ `0 R2 ^. J
8 G, Z1 v7 Y1 i8 T5 f- w, g ' [- t' P* ^! A Q0 }- G/ o; G% z$ ?: w
Matter K, Hunziker W, and Mellman I. Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell 71: 741-753, 1992. " @7 ]" z& l( O9 l7 D" j ! S0 W; r$ Q) M6 f3 Q E( I$ `/ `. B, `' y( k& B" I( ?5 W
3 @- J8 T5 L, _3 W; ~) O
Pessia M, Imbrici P, D'Adamo MC, Salvatore L, and Tucker SJ. Differential pH sensitivity of Kir4.1 and Kir42 potassium channels and their modulation by heteropolymerisation with Kir51. J Physiol 532: 359-367, 2001. 6 l E9 L' ^) l- q) _4 Z/ g 7 V+ F0 G7 [+ Z4 I/ t5 |* n) E1 D* D4 }- X4 W E
% @3 {2 i- J1 x8 D' z
Pessia M, Tucker SJ, Lee K, Bond CT, and Adelman JP. Subunit positional effects revealed by novel heteromeric inwardly rectifying K channels. EMBO J 15: 2980-2987, 1996.3 v: T1 z) [) Y1 y" z7 s
/ \4 I0 P) R! b& S' i# ~' S) r9 y" ~
4 G& j, r4 Y ]4 U 4 L+ y/ M& d0 V- `7 V0 b' p" [Sheng M. Molecular organization of the postsynaptic specialization. Proc Natl Acad Sci USA 98: 7058-7061, 2001.9 V- a1 C( M3 O s8 k3 h7 d
) K# s h8 O* u5 L0 d1 ?3 T: c3 q1 Q& h4 K; T2 H
# ^9 L; d6 e5 x" J( IShuck ME, Piser TM, Bock JH, Slightom JL, Lee KS, and Bienkowski MJ. Cloning and characterization of two K inward rectifier (Kir) 1.1 potassium channel homologs from human kidney (Kir12 and Kir13). J Biol Chem 272: 586-593, 1997. * p3 G" H- s$ n. l: x+ B2 y3 ]/ T! V) Z8 K' Z) R
" b9 S7 R* O0 q9 `4 {# N9 A. [3 }! l2 p% a4 W5 G* T& y- l g' k
Simon DB, Karet FE, Rodriguez-Soriano J, Hamdan JH, DiPietro A, Trachtman H, Sanjad SA, and Lifton RP. Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K channel, ROMK. Nat Genet 14: 152-156, 1996.6 M7 E7 V' E/ `, Z* P% V7 I
( q+ h' L/ [' @8 N3 A3 Q4 U6 {, Q A- v: u/ A- P
9 r% G7 |) U4 {3 T
Tanemoto M, Fujita A, Higashi K, and Kurachi Y. PSD-95 mediates formation of a functional homomeric Kir5.1 channel in the brain. Neuron 34: 387-397, 2002." f8 }5 h9 F5 s# R4 e
t9 x6 C. Q3 P7 ]6 u4 N. l5 X
$ d; @4 @( ?4 D; R7 L- ]: k, F* } # ~7 n$ D. {5 L9 b. g: `, N; lTanemoto M, Kittaka N, Inanobe A, and Kurachi Y. In vivo formation of a proton-sensitive K channel by heteromeric subunit assembly of Kir5.1 with Kir41. J Physiol 525: 587-592, 2000.) D% }/ C7 Q, {
, ?1 ]' `; T5 p' T, c8 M9 L" E 0 d$ [: C9 [" z: i! D' S t. F) J3 w" t/ ^/ P
Tanemoto M, Vanoye CG, Dong K, Welch R, Abe T, Hebert SC, and Xu JZ. Rat homolog of sulfonylurea receptor 2B determines glibenclamide sensitivity of ROMK2 in Xenopus laevis oocyte. Am J Physiol Renal Physiol 278: F659-F666, 2000.3 D$ i, P7 t0 v, i6 y% ?3 D& o1 |
, i1 X$ Q; s' p6 W5 D% }
9 K: p# j' h9 X, q5 T$ d! R, F% w6 w$ A& ]: L1 ?& y C' Q, t8 n) f" N- m
Tojo A, Bredt DS, and Wilcox CS. Distribution of postsynaptic density proteins in rat kidney: relationship to neuronal nitric oxide synthase. Kidney Int 55: 1384-1394, 1999. 5 S/ L6 D6 y0 m$ y" _% }! q% Z+ j H. ?- T9 _' U( ^
& a3 {% k( h9 |8 }3 b0 E: B ; h) n: z/ S6 X8 K1 x, Y& JTucker SJ, Imbrici P, Salvatore L, D'Adamo MC, and Pessia M. pH dependence of the inwardly rectifying potassium channel, Kir5.1, and localization in renal tubular epithelia. J Biol Chem 275: 16404-16407, 2000.. k! D. {: ]; y0 Z
/ P7 h+ o+ s5 t2 Y: S7 c; N, M9 `+ F! r! K. k% q
/ N# ]& r3 P' ^" I$ Y2 D' j
Xu H, Cui N, Yang Z, Qu Z, and Jiang C. Modulation of kir4.1 and kir5.1 by hypercapnia and intracellular acidosis. J Physiol 524: 725-735, 2000. , z y- z5 E3 Y( F3 R0 U( A2 C+ ~% L5 T# d
& Y- E) _7 v: J0 a
% _; ?$ e: m9 e
Xu JZ, Hall AE, Peterson LN, Bienkowski MJ, Eessalu TE, and Hebert SC. Localization of the ROMK protein on apical membranes of rat kidney nephron segments. Am J Physiol Renal Physiol 273: F739-F748, 1997.- z2 q% d9 G2 ]' e0 u- V
5 l) A+ w/ J) D: w
# `. m3 W$ a6 |" {% A2 x
. f! l* G6 e& u) nYeaman C, Grindstaff KK, and Nelson WJ. New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol Rev 79: 73-98, 1999.作者: 泡泡鱼 时间: 2015-6-13 20:10