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Originally published In Press as doi:10.1074/jbc.M405985200 on August 13, 2004

J. Biol. Chem., Vol. 279, Issue 42, 44065-44073, October 15, 2004
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Differential Assembly of Inwardly Rectifying K+ Channel Subunits, Kir4.1 and Kir5.1, in Brain Astrocytes*

Hiroshi Hibino{ddagger}, Akikazu Fujita§, Kaori Iwai{ddagger}, Mitsuhiko Yamada{ddagger}, and Yoshihisa Kurachi{ddagger}

From the {ddagger}Department of Pharmacology II, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan and §Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan

Received for publication, May 28, 2004 , and in revised form, August 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The inwardly rectifying K+ channel subunit Kir5.1 is expressed abundantly in the brain, but its precise distribution and function are still largely unknown. Because Kir5.1 is co-expressed with Kir4.1 in retinal glial Müller cells, we have compared the biochemical and immunological properties of Kir5.1 and Kir4.1 in the mouse brain. Immunoprecipitation experiments suggested that brain expressed at least two subsets of Kir channels, heteromeric Kir4.1/5.1 and homomeric Kir4.1. Immunolabeling using specific antibodies showed that channels comprising Kir4.1 and Kir5.1 subunits were assembled in a region-specific fashion. Heteromeric Kir4.1/5.1 was identified in the neocortex and in the glomeruli of the olfactory bulb. Homomeric Kir4.1 was confined to the hippocampus and the thalamus. Homomeric Kir5.1 was not identified. Kir4.1/5.1 and Kir4.1 expression appeared to occur only in astrocytes, specifically in the membrane domains facing the pia mater and blood vessels or in the processes surrounding synapses. Both Kir4.1/5.1 and Kir4.1 could be associated with PDZ domain-containing syntrophins, which might be involved in the subcellular targeting of these astrocyte Kir channels. Because heteromeric Kir4.1/5.1 and homomeric Kir4.1 have distinct ion channel properties (Tanemoto, M., Kittaka, N., Inanobe, A., and Kurachi, Y. (2000) J. Physiol. (Lond.) 525, 587-592 and Tucker, S. J., Imbrici, P., Salvatore, L., D'Adamo, M. C., and Pessia, M. (2000) J. Biol. Chem. 275, 16404-16407), it is plausible that these channels play differential physiological roles in the K+-buffering action of brain astrocytes in a region-specific manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glial cells are highly specialized support cells in the nervous system. They exert their functions by closely associating with neuronal synapses, axons, dendrites, and soma (3). Glial cell processes can also reach out to more distant regions such as blood vessels, the pia mater, and the vitreous humor. Astrocytes are a particular subset of glial cells whose primary function lies in the control of the ionic and osmotic composition of the extracellular environment. Physiological analyses have revealed that astrocytes possess a large K+ conductance (4, 5) that is thought to be involved in the polarized transport of K+ from regions of high extracellular K+ ([K+]o) to those of low [K+]o. This "spatial buffering of K+," which is also known as "K+-siphoning" when applied to retinal astrocytes (Müller cells), is thought to be essential for the maintenance of neuronal activity as it prevents accumulation of excess [K+]o, which would otherwise interfere with neuronal excitability (5, 6). Because an inwardly rectifying K+ (Kir) channel subunit, Kir4.1, and a water channel, AQP4, are found closely co-localized on specific membrane domains of astrocytes and Müller cells, it is assumed that the K+ buffering is also essential for neural osmotic homeostasis by coupling with water movement (7-13).

It has been proposed that Kir4.1 is a major component of the K+ conductance in brain astrocytes and retinal Müller cells (14-17). A recent immunohistochemical study of the retina has revealed that Müller cells express not only homomeric Kir4.1 but also heteromeric Kir4.1/5.1 channels (18). The two Kir channels distributed differentially within a Müller cell. They also have different biophysical properties and intracellular pH (pHi) dependence (1, 2, 19). It is, therefore, suggested that the two Kir channels play distinct functional roles in the buffering of K+; the homomeric Kir4.1 localized at end feet facing the vitreous surface and at processes wrapping blood vessels may allow K+ extrusion, whereas heteromeric Kir4.1/5.1 channels at processes surrounding neurons may be responsible for pHi-dependent K+ uptake. Although it was shown that Kir5.1 subunit was abundantly expressed in the brain as well (20, 21), little is known about its role in formation of brain Kir channel and its precise distribution.

In the present study we compared the biochemical and immunological properties of brain Kir5.1 and Kir4.1. We found that the Kir subunits form at least two types of channels, heteromeric Kir4.1/5.1 and homomeric Kir4.1, in the brain. Both Kir channels were expressed in astrocytes but not in neurons. Within an astrocyte, each type of Kir channel distributed to specific membrane domains, i.e. perivascular or perisynaptic area, in a region-specific manner. We also found that Kir4.1/5.1 and Kir4.1 channels could bind syntrophins, which are components of the dystrophin-associated protein complex (DAPC).1 Because the DAPC has been proposed to be responsible for the polarized distribution of Kir4.1 in Müller cells (22, 23), syntrophins may be at least partially involved in the targeting of the Kir channels to a specific membrane domain in astrocytes. Taken together our results suggest that the assembly of Kir5.1 with Kir4.1 conveys particular properties to the K+-buffering action of astrocytes in various areas of the brain.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Generation of specific antibodies against Kir4.1 (KAB-2C2) and Kir5.1 (Kir5.1-NR1) was as previously described (18, 24). Antibodies specific for Kir4.2 (epitope; residues 347-366 of mouse Kir4.2, GenBankTM accession number O88932 [GenBank] ) and AQP4 (epitope; residues 249-323 of rat AQP4, GenBankTM accession number P47863 [GenBank] ) were purchased from Alomone Laboratories (Jerusalem, Israel).

Preparation of Membrane Fractions and Western Blotting—Membrane fractions of kidney and brain were prepared as described elsewhere (25). Briefly, whole brain and kidney cortex of adult female C57BL/6 mice (7~8 weeks old) were homogenized in 9 volumes of dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2) containing a protease inhibitor mixture (Roche Applied Science). The homogenate was centrifuged at 4000 x g for 15 min to remove nuclei, mitochondria, and any remaining large cellular fragments. The supernatant was further centrifuged at 17,000 x g for 30 min. The pellet was harvested as a membrane fraction. The membrane protein was then heated for 10 min at 70 °C in the sample buffer containing {beta}-mercaptoethanol and analyzed by SDS-PAGE and immunoblotting with anti-Kir4.1, anti-Kir4.2, or anti-Kir5.1 antibody.

Deglycosylation and Inhibition of Glycosylation—Extracellular glycocalyx moieties were removed from brain and renal membrane proteins (deglycosylation) by dilution of 40 µg of protein in a buffer containing 20 mM sodium phosphate, 10 mM EDTA, and 0.5% Nonidet P40, pH 7.2, and incubated with N-glycosidase F (Roche Applied Science) for 17 h at 37 °C. The proteins were directly analyzed using Western blotting.

In some cases glycosylation of transiently expressed Kir4.1 and Kir4.2 protein (HEK293T) was inhibited by supplementing the culture medium with 1.5 µg/ml tunicamycin (Sigma) for 15 h. The cells were harvested, lysed by a solution containing 1% Triton X-100, 0.5% deoxycholic acid, and 150 mM NaCl in 40 mM Tris-HCl at pH 7.4, and finally analyzed by Western blotting.

Tissue Culture and Immunoprecipitation—Using LipofectAMINE (Invitrogen) according to the manufacturer's protocol, we transiently transfected HEK293T cells with full-length cDNA of either Kir4.1 or Kir5.1 inserted into the mammalian expression vectors pCMV-HA (Clontech, Palo Alto, CA) or pcDNA3.1(+) (Invitrogen), respectively. Some experiments employed simultaneous co-expression of an Myc-tagged construct of full-length {alpha}-syntrophin inserted into pCMV-Myc (Clontech), which had been obtained by reverse transcription-PCR from mouse brain. Interaction between heteromeric Kir4.1, Kir5.1, and {alpha}-syntrophin was assessed by co-transfection of all three constructs. The cells were harvested 48 h after the transfection, and immunoprecipitation assays were conducted as previously described (26). Briefly, after extraction with a lysis solution containing 1% Triton X-100, 0.5% deoxycholic acid, and 150 mM NaCl in 40 mM Tris-HCl at pH 7.4, the proteins were incubated overnight at 4 °C with 30 µl of anti-Myc antibody-conjugated beads (Clontech). Subsequently, the beads were washed four times with the above solution and spun down, and co-precipitated proteins were resolved by SDS-PAGE and probed with anti-Kir4.1, anit-Kir5.1, or anti-Myc antibody. Immunoprecipitation assays using native tissues were done by extracting the membrane fraction from brain and kidney with lysis solution followed by precipitation of A/G-agarose beads (30 µl) (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated to preimmune IgG, anti-Kir4.1, or anti-Kir5.1 antibody (0.6 µg each) with 500 µg of solubilized protein.

Glutathione S-Transferase (GST) Pull-down Assays—DNA fragments corresponding to PDZ domains of {alpha} (amino acid residues 70-188, GenBankTM accession number U00677 [GenBank] ), {beta}1 (81-220, accession number U89997 [GenBank] ), {beta}2 (51-208, accession number U00678 [GenBank] ), and {gamma}1 (41-177, accession number AF367759 [GenBank] ) syntrophins were amplified via reverse transcription-PCR from mouse brain and subcloned into the pGEX-4T-1 vector (Amersham Biosciences). The BL21 strain of Escherichia coli was transformed with the vector constructs, and protein expression was induced by the addition of 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside for 3-4 h at 37 °C. Bacteria were sonicated in lysis buffer and centrifuged, the lysates were recovered, and the fusion proteins were purified on glutathione-Sepharose.

Pull-down assays were done as previously described (27). HEK293T cells transfected with GFP-tagged Kir4.1, Kir5.1, or Kir4.1 plus Kir5.1 (in pEGFP, Clontech) were solubilized in lysis buffer and centrifuged at 100,000 x g for 15 min at 4 °C. One hundred micrograms of solubilized proteins were incubated overnight at 4 °C with 30 µl of glutathione-Sepharose beads bound to 5 µg of purified fusion protein. Samples were washed 4 times at room temperature with the same buffer. The materials retained on the beads were eluted with sample-buffer solution and analyzed by SDS-PAGE and immunoblotting using anti-GFP or anti-Kir5.1 antibody.

Immunohistochemistry—All of the brain samples for immunohistochemistry were prepared as previously described (28, 29). Briefly, the bodies of adult female C57BL/6 mice, which were deeply anesthetized with pentobarbital sodium (100 mg/kg), were perfused from their left ventricle with 4% paraformaldehyde, 0.1 M sodium phosphate, pH 7.4, and the brains were isolated. The procedures for double-immunolabeling of slice sections have been previously published (14, 16, 30, 31). In short, cryosections of brain (12 µm) were at first incubated with anti-Kir5.1 (4 µg/ml; Figs. 2 and 3A) or anti-Kir4.1 (0.3 µg/ml; Fig. 3A), then washed three times with phosphate-buffered saline and treated with fluorescein-conjugated goat anti-rabbit IgG (Fab fragment) secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Subsequent to another phosphate-buffered saline wash, either anti-Kir4.1 (0.3 µg/ml; Fig. 2) or anti-AQP4 (0.2 µg/ml; Fig. 3A) was applied to the brain sections. The sections were washed again with phosphate-buffered saline and finally treated with Texas Red-labeled anti-rabbit IgG secondary antibody. The samples were examined under an LSM-510 confocal microscope (Zeiss, Jena, Germany). To confirm the specificity of the double-labeling, we incubated the sections with IgG derived from rabbit preimmune serum instead of anti-Kir4.1 or AQP4 antibody and observed no fluorescence of Texas Red in the sections (data not shown).



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  		FIG. 2.
Expression of Kir4.1 and Kir5.1 in various regions of the mouse brain. A-D, mouse brain sections were probed with anti-Kir4.1 and anti-Kir5.1 antibody and labeled by Texas Red (red) and fluorescein-conjugated (green) secondary antibody, respectively. Duplicate images of the same region displaying either Kir4.1- or Kir5.1-specific labeling were merged to visually emphasize co-localization (yellow) of the proteins. A, neocortex. Arrowheads and arrows delineate immunolabeling deep to the pia mater (P) and around blood vessels (BV), respectively. B, glomerular (G) layer of the olfactory bulb. Arrowheads mark extraglomerular regions where Kir4.1 labeling (red) was predominant. C, hippocampus. D, LPMR, the mediorostral area of lateral post-thalamic nucleus. Pr, pyramidal cell layer; St, stratum radiatum.

 



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  		FIG. 3.
Distribution and localization of Kir4.1 and Kir5.1 in the neocortex. A, double-immunolabeling with anti-AQP4 (red) and either Kir4.1 or Kir5.1 indicated co-localization of both Kir subunits with AQP4 in astrocyte processes attaching to the pia mater (arrowheads) or wrapping around blood vessels (arrows). Note that Kir labeling is green in both panels (i.e. Kir4.1 and Kir5.1). BV, blood vessels; P, pia mater. B, a-c, neocortical localization of Kir4.1 and Kir5.1 analyzed by immunoelectron microscopy. Sections were pre-embedded as outlined under "Experimental Procedures" and examined for immunoreactivity highlighting either Kir4.1 (left) or Kir5.1 (right). Notable signal for both subunits was detected on the membrane of astrocyte processes around blood vessels (Ba) as well as those surrounding type I (Bb) and II (Bc) synapses. Refer to Table I for numerical data. End, endothelial cells; asterisks, postsynaptic side; triangles, presynaptic side.

 
Immunoelectron Microscopic Examination—Pre-embedding immunoelectron microscopic analysis of mouse brain with either Kir4.1 or Kir5.1 antibody was performed as described previously (16). Anti-Kir4.1 (6 µg/ml) or anti-Kir5.1 (40 µg/ml) was used to examine thin sections (thickness, 75~90 nm) of mouse brain neocortex. Resulting immunoreactivity was visualized by the Vectastain Elite ABC kit (Vector laboratories, Burlingame, CA) and 3,3'-diaminobenzidine.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Biochemical Characterization of Brain Kir4.1 and Kir5.1—We first examined the expression of Kir4.1 and Kir5.1 in the mouse brain using antibodies of established specificity (18, 24). In some experiments we also examined the renal expression of Kir4.1 and Kir5.1 for comparison (1, 2, 32).

In brain preparations the anti-Kir4.1 antibody visualized four protein bands at ~40, ~80, ~160, and ~250 kDa in the SDS-PAGE (Fig. 1Aa, first lane). The molecular mass of the lowest band (~40 kDa) was almost identical to the predicted mass of the Kir4.1 subunit based on its cDNA length (~42 kDa), suggesting that this band represented monomeric Kir4.1. Kir channels are tetrameric, and it is likely that the ~80- and ~160-kDa fragments correspond to complexes of 2 and 4 Kir subunits that contain at least one Kir4.1 subunit. The heaviest fragment (~250 kDa) may represent a macro protein complex containing Kir4.1 (17, 22). A slightly different pattern was found in human embryonic kidney (HEK293T) cells transfected with Kir4.1 cDNA (Fig. 1Ad). In addition to the bands at ~80, ~160, and ~250 kDa (data not shown), two bands appeared at ~33 and ~38 kDa (Fig. 1Ad, first lane) rather than at ~40 kDa, corresponding to the putative brain Kir4.1 monomer (Fig. 1Ad, third lane). Kir4.1 possesses an N-glycosylation motif in its extracellular domain (33), and the degree of glycosylation may alter the mobility of the protein on gels. Preincubation of the transfected cells with tunicamycin, a glycosylation inhibitor, abolished the ~38-kDa fragment and enhanced the band at ~33 kDa (Fig. 1Ad, second lane). Treatment of brain protein fractions with a deglycosylation agent, N-glycosidase, shifted some of the ~40-kDa fragment to ~33 kDa (Fig. 1Ad, fourth lane). We, therefore, conclude that the band at ~40 kDa in the brain preparation and the ~38-kDa band from Kir4.1-transfected HEK293T cells represent monomeric Kir4.1 subunits. The deglycosylated Kir4.1 monomers showed faster electrophoretic mobility (~33 kDa) than that predicted from its molecular mass (~42 kDa).



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  		FIG. 1.
Biochemical characterization of Kir4.1, Kir5.1, and Kir4.2 proteins in mouse brain and kidney. A, a-c, Western blot analyses of membrane fractions from untreated lysates of brain (A, a-c, first lane) and renal cortex (Aa, third lane; Ab-Ac, second lane) or those treated with N-glycosidase (Aa, second and fourth lanes). The proteins were loaded and immunoblotted (IB) with the antibody indicated at the bottom of the panels. A, d-e, Western blot analyses of Kir subunits transiently expressed (HEK293T, first and second lanes) or isolated from native tissues (third and fourth lanes; d, brain; e, kidney). Treatment with tunicamycin (second lanes) or N-glycosidase (fourth lanes) is indicated at the top. The type of antibody used during the immunoblotting is denoted at the bottom. IP, immunoprecipitation. B, a-b, co-immunoprecipitation of Kir4.1 and Kir5.1 from brain. A solubilized membrane fraction from brain was immunoprecipitated by preimmune IgG (second lane), anti-Kir4.1 (third lane), or anti-Kir5.1 (fourth lane), and the precipitants were probed with anti-Kir4.1 (Ba) or anti-Kir5.1 (Bb) antibody. Anti-Kir5.1 and anti-Kir4.1 antibodies precipitated Kir4.1 and Kir5.1 proteins, respectively (wide solid arrowheads) (Ba, fourth lane; Bb, third lane), indicating a heteromeric assembly of both subunits in the brain. Note that immunoreactivity at ~80, ~160, and ~250 kDa was detected only when the anti-Kir4.1-precipitated protein was probed with the same antibody (Ba, third lane). Open arrowheads point at the heavy chain of IgG.

 
Control experiments conducted in the kidney showed that the anti-Kir4.1 antibody detected several fragments at ~50-kDa, but no bands corresponding to the multimers were observed in the brain (Fig. 1Aa, third lane). Glycosylation inhibitors reduced Kir4.1 immunoreactivity in the kidney from ~50 to ~33 kDa (Fig. 1Aa, fourth lane). Glycosylation of Kir4.1 was, thus, notably higher in the kidney.

On the other hand, immunoanalysis of Kir5.1 protein in the brain and kidney yielded only one clear band at ~36 kDa (Fig. 1Ab). This was considerably smaller than the estimated size of Kir5.1 based on its cDNA length (~46 kDa). Similarly, we found only one band at ~36 kDa in preparations from HEK293T cells transfected with Kir5.1 cDNA (data not shown).

The Kir4.2 channel subunit is known to couple with Kir5.1 in the kidney (Fig. 1Ac, second lane) (34, 35). But in the brain we detected no significant signal for Kir4.2 protein (Fig. 1Ac, first lane), although its mRNA has been reported to be expressed (36). On SDS-PAGE the Kir4.2 protein expressed in HEK293T cells was detected at ~37 and ~35 kDa and in kidney at ~45, ~37, and ~35 kDa (Fig. 1Ae, first and third lanes, respectively). Like Kir4.1, Kir4.2 possesses a glycosylation motif (34, 37). In the presence of glycosylation inhibitors, Kir4.2 proteins were represented by a single band at ~35 kDa in both cases (Fig. 1Ae, second and fourth lanes).

Assembly of Kir4.1 and Kir5.1 has been reported to yield functional Kir channels in renal epithelia (1, 2, 32) and retinal Müller cells (18). To see if such heteromeric assembly occurred in the brain, we performed immunoprecipitation with solubilized brain lysate using the antibodies for Kir4.1 or Kir5.1 (Figs. 1B, a and b). The immunoprecipitants with anti-Kir5.1 contained Kir4.1-immunoreactivity (~40 kDa; Fig. 1Ba, fourth lane, wide solid arrowhead), and those precipitated with anti-Kir4.1 contained Kir5.1 (~36 kDa; Fig. 1Bb, third lane, wide solid arrowhead). This data suggests that the brain expresses Kir4.1/5.1 heteromers but in neither set of immunoprecipitates did cross-reaction of Kir4.1 and Kir5.1 antibodies occur at anything other than the monomer protein level. Although in the precipitants with anti-Kir4.1 antibody the same antibody detected protein bands corresponding to dimers (~80 kDa) and tetramers (~160 kDa) (Fig. 1Ba, third lane), these were not recognized by the anti-Kir5.1 antibody (Fig. 1Bb, third lane). Immunoreactivity for Kir5.1 in the Kir5.1-antibody precipitants could only be detected at gel positions consistent with monomeric Kir5.1 (Fig. 1Bb, fourth lane). This single band was also recognized in the precipitants with the anti-Kir4.1 antibody when they were probed with anti-Kir5.1 antibody (Fig. 1Bb, third lane, wide solid arrowhead). No Kir4.1 immunoreactivity at ~80 or ~160 kDa was detected in the immunoprecipitants with anti-Kir5.1 antibody (Fig. 1Ba, fourth lane). It, therefore, seems that in the brain neither homomeric Kir5.1 nor heteromeric Kir4.1/5.1 channels maintain their subunit association during SDS-PAGE analysis. On the other hand, the homomeric interaction between Kir4.1 subunits is preserved. Taken together these observations imply that Kir4.1 and Kir5.1 form at least two sets of functional Kir channels in the brain, heteromeric Kir4.1/5.1 and homomeric Kir4.1, although on this basis we cannot exclude the possibility of homomeric Kir5.1 channels.

Distribution of Kir4.1 and Kir5.1 Proteins in the Brain—Double-immunolabeling showed that Kir5.1 co-localized with astrocyte-specific glial fibrillary acidic protein but not with neuron-specific enolase in the neocortex (data not shown), which suggests that, like Kir4.1 (16), Kir5.1 is expressed exclusively in astrocytes and not in neurons. Kir4.1 is abundantly expressed in astrocytes of many brain regions (16, 17). We, therefore, compared the distribution of Kir5.1 in mouse brain with that of Kir4.1 by double-immunolabeling (Fig. 2).

Strong labeling for Kir4.1 (red) and Kir5.1 (green) was detected in the neocortex (Fig. 2A), with notable overlaps in various regions (yellow), especially around the blood vessels (arrows) as well as deep in the pia mater (arrowheads). Co-localization of Kir4.1 and Kir5.1 was also detected in astrocytes of other regions, such as the pontine nucleus and the cranial nerve nuclei of the hindbrain (data not shown). Double-immunolabeling with anti-Kir4.1 and anti-Kir5.1 antibodies produced yellow signals on the inside and at the edges of the olfactory glomeruli (Fig. 2B). Careful examination of the images revealed that the outside of the glomeruli (arrowheads) was primarily labeled by anti-Kir4.1, with only a weak signal for Kir5.1.

We next examined the CA1 region of the hippocampus (Fig. 2C). Kir4.1 was moderately expressed in the processes of astrocytes in the pyramidal cell layer and the stratum radiatum (16, 17). Relatively weak labeling for Kir5.1 was scattered in or close to the pyramidal cell layer, where it was largely co-localized with the Kir4.1-specific signal. In stratum radiatum, although little Kir5.1-immunoreactivity was detected, Kir4.1 and Kir5.1 labeling again appeared to be co-localized around blood vessels. In the mediorostral region of the lateral post-thalamic nucleus (Fig. 2D), we only detected labeling for Kir4.1 and not for Kir5.1.

The expression of AQP4 in the brain is restricted to astrocyte membranes facing blood vessels or synaptic terminals (13, 38). We compared immunolabeling for either Kir4.1 or Kir5.1 with a specific antibody for AQP4 (Fig. 3A) in the neocortex where Kir4.1 and Kir5.1 subunits were abundantly expressed (see Fig. 2A). Immunolabeling for AQP4 (red) was closely associated with the pial surface (arrowheads) and blood vessels (arrows). Double-labeling of AQP4 and either anti-Kir4.1 or anti-Kir5.1 antibody (Fig. 3A, left and right, respectively, both green) gave rise to intensive yellow labeling in both cases. This suggests the co-localization of heteromeric Kir4.1/5.1 and AQP4 in the processes of these astrocytes.

Localization of Kir4.1 and Kir5.1 in Astrocytes—The localization of Kir5.1 and Kir4.1 in astrocytes of the mouse neocortex was examined by immunoelectron microscopy (Figs. 3B, a-c). Immunolabeling for Kir4.1 was prominently detected in the perivascular processes of astrocytes (Fig. 3Ba, left). The pattern of Kir5.1 labeling was similar to that of Kir4.1 (Fig. 3Ba, right). Immunolabeling for both Kir4.1 and Kir5.1 was evident in all perivascular processes we examined (Kir4.1, n = 16; Kir5.1, n = 17), which suggests that Kir channels in the vascular processes of astrocytes are composed of heteromeric Kir4.1/5.1 subunits.

We detected Kir4.1 and Kir5.1 labeling (Figs. 3B, b and c, left and right, respectively) in the processes of astrocytes surrounding both type I (Fig. 3, Bb) and type II (Fig. 3, Bc) synapses (39). Approximately 80% of type I synapses and 50% of type II synapses were surrounded by Kir4.1-positive processes (Table I). Kir5.1-immunopositive processes surrounded more than 50% of both type I and type II synapses. Our findings suggest that synaptic connections in the neocortex are surrounded by astrocyte processes expressing at least two channels, homomeric Kir4.1 and heteromeric Kir4.1/5.1.


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  		TABLE I
Incidence of Kir4.1 and Kir5.1 expression in astrocytes at excitatory and inhibitory synapses of the mouse neocortex

 
PDZ Domain-mediated Interaction of Kir4.1 and Kir4.1/5.1 with Syntrophins—It is clear that homomeric Kir4.1 and heteromeric Kir4.1/5.1 channels are distributed in specific membrane domains of brain astrocytes. A similar situation exists in retinal Müller cells (14, 18, 40), where the DAPC has been suggested to play a key role in the selective and specific localization of Kir4.1 (22, 41). Both Kir4.1 and Kir5.1 possess a PDZ (PSD-95-Disc large-ZO1) domain binding motif at their carboxyl-terminal end and can physically bind to PSD-95 family proteins (21, 42). Among the proteins incorporated in the DAPC, only syntrophins contain a PDZ domain, and {alpha}-syntrophin may interact with Kir4.1 in brain astrocytes (23). We, therefore, tested whether Kir4.1 and Kir5.1 could associate with syntrophins (Fig. 4).



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  		FIG. 4.
Homomeric Kir4.1 and heteromeric Kir4.1/5.1 channels can associate with syntrophins. Aa, Kir4.1, but not Kir5.1, interacts with GST fusion proteins containing PDZ domains of {alpha} (third lane), {beta}1 (fourth lane), and {beta}2 (fifth lane) syntrophins. Lysates from transiently transfected cells (GFP-Kir4.1 or GFP-Kir5.1) were subjected to GST pull down and blotted with GFP antibody. Note that Kir4.1 could not bind the PDZ domain of {gamma}1-syntrophin (sixth lane). Controls, Kir4.1 and Kir5.1, both bound to a fusion protein containing PDZ2, PDZ3, SH3, and GK domains of PSD-95 (seventh lane) but not to a similar fusion protein containing only the SH3 and GK domains (eighth lane). A, b-c, interaction of {alpha}-syntrophin with Kir4.1 and Kir5.1. HEK293T cells were transfected with either Kir4.1 (Ab) or Kir5.1 (Ac) alone (second lanes) or in co-transfection with a Myc-tagged construct of full-length {alpha}-syntrophin (third lanes). Immunoprecipitation (IP) was conducted with anti-Myc antibody and subsequent immunoblotting (IB) with anti-Kir4.1 (Ab, top), anti-Kir5.1 (Ac, top), or anti-Myc (Ab-Ac, bottom). Only Kir4.1, but not Kir5.1 could be co-precipitated in double-transfection setups (third lanes). Neither Kir4.1 nor Kir5.1 when expressed alone was precipitated by the antibody (second lanes). The gray arrowhead in Ab (top) points at unglycosylated Kir4.1 protein. The open arrowhead marks the heavy chain of IgG. B, the Kir4.1 carboxyl terminus is crucial for syntrophin interaction. GFP-tagged Kir4.1 lacking the last three amino acid residues at the carboxyl-terminal end (GFP-Kir4.1{Delta}C3) no longer bound to the {alpha} or {beta}1-syntrophin GST-fusion proteins described in panel Aa (anti-GFP, third and fourth lanes) but could still be co-precipitated with Kir5.1 by Kir5.1-specific antibody (fifth lane). All analyses were done on transiently transfected cells. The proteins were visualized by anti-GFP antibody. The open arrowhead marks heavy chain IgG. C, a-b, Kir5.1-syntrophin interaction requires Kir4.1 co-expression. Ca, GFP-tagged Kir5.1 was co-expressed with GFP-tagged Kir4.1 in HEK293T cells, and the cell lysates were analyzed with the syntrophin GST fusion proteins described in panel (Aa). GST-mediated pull-down and immunoblotting with anti-Kir5.1 revealed the presence of GFP-Kir5.1 in the precipitated protein complex (third lane, {alpha}-syntrophin: fourth lane, {beta}1-syntrophin). Control experiments verifying PDZ binding were identical to panel Aa. Cb, HEK293T cells were transfected with the constructs of HA-tagged Kir4.1, Kir5.1, and Myc-tagged full-length {alpha}-syntrophin ({alpha}-Synt), and the cell lysates were precipitated by anti-Myc antibody. The membranes were probed with anti-Kir4.1 (top), anti-Kir5.1 (middle), or anti-Myc (bottom) antibody. The gray arrowhead in Cb (top) indicates unglycosylated Kir4.1 protein. The open arrowhead marks heavy chain IgG.

 
Glutathione beads charged with GST fusion proteins containing the PDZ domain of {alpha}, {beta}1, {beta}2, or {gamma}1 syntrophin were incubated with lysates of HEK293T cells expressing GFP-tagged constructs of Kir4.1 or Kir5.1 (Fig. 4A). Bound proteins were analyzed by Western blotting with anti-GFP antibody. Kir4.1 subunits interacted with the PDZ domains of all but one ({gamma}1) of the syntrophins tested (Fig. 4Aa, top). Kir5.1, by contrast, failed to bind any of the syntrophin constructs (Fig. 4Ab, bottom) despite its known ability to associate with the PDZ domain of PSD-95 (21).

We next examined whether the Kir subunits could bind to full-length {alpha}-syntrophin. HEK293T cells were transiently transfected with either Kir4.1 or Kir5.1 in the presence or absence of Myc-tagged full-length {alpha}-syntrophin, and the cell lysates were used for immunoprecipitation (Figs. 4A, b and c). Anti-Myc antibodies precipitated Kir4.1 when it was co-expressed with Myc-syntrophin (Fig. 4Ab, third lane) but failed to do so from lysates of cells expressing Kir4.1 alone (Fig. 4Ab, second lane). Kir5.1 could not be precipitated by the anti-Myc antibody even when co-expressed with Myc-syntrophin (Fig. 4Ac, third lane). These results demonstrate that Kir4.1, but not Kir5.1, could interact with full-length {alpha}-syntrophin.

We next attempted to map the syntrophin interaction site within Kir4.1 (Fig. 4B). The GST-syntrophin fusion proteins mentioned above were incubated with lysates of HEK293T cells transfected with a GFP-Kir4.1 construct lacking the last three carboxyl-terminal amino acids (GFP-Kir4.1{Delta}C3). None of the tested syntrophin PDZ domains precipitated GFP-Kir4.1{Delta}C3 (Fig. 4B, third and fourth lanes). Thus, Kir4.1 interacts with syntrophins through its carboxyl-terminal PDZ domain binding motif (amino acid residues 377-379, Ser-Asn-Val, GenBankTM accession number NM_002241 [GenBank] ).

However, GFP-Kir4.1{Delta}C3 could still assemble with the Kir5.1 subunit because it could be immunoprecipitated by anti-Kir5.1 antibody (Fig. 4B, fifth lane), and pull-down of syntrophin GST fusion proteins showed that co-expression of GFP-Kir4.1 and GFP-Kir5.1 resulted in complexing of Kir5.1 with syntrophin (Fig. 4Ca, third and fourth lanes, Kir5.1-specific antibody). By the same token immunoprecipitation of Mycsyntrophin using lysates from the cells co-expressing Kir4.1 and Kir5.1 confirmed that all three proteins were in the same complex (Fig. 4Cb, third lane). Therefore, homomeric Kir4.1 and heteromeric Kir4.1/5.1 channels can associate with {alpha}- and {beta}-syntrophins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Heteromeric Kir4.1/5.1 and Homomeric Kir4.1; Distinct Channel Localization Argues for Physiological Relevance—This study shows that Kir5.1 forms a functional Kir channel with Kir4.1 in brain astrocytes that is not expressed in neurons (Figs. 2 and 3). The distribution of Kir5.1 immunoreactivity is largely consistent with that of its mRNA (20), although the mRNA was found also in some neurons. Homomeric assembly of Kir5.1 was not confirmed in this study, whereas homomeric Kir4.1 channels were identified. Therefore, the glial K+ conductance, which is responsible for K+ buffering in the brain, is mainly composed of two types of Kir channels, homomeric Kir4.1 and heteromeric Kir4.1/5.1. These Kir channels, which are differentially distributed in the brain (see Fig. 2), exhibit distinct electrophysiological properties (1, 2); 1) the single-channel conductance of the Kir4.1/5.1-heteromer is larger than that of the Kir4.1-homomer, 2) the heteromer shows burst-like activity, whereas the homomer appears to be continuously open with brief interruptions, and 3) the activity of the heteromer is much more sensitive to intracellular pH (pHi) in the physiological range than the homomer. It is, therefore, suggested that these two channels play functionally different roles in glial K+-buffering in the brain.

It is particularly important for the function of the glial Kir channels that heteromeric Kir4.1/5.1 channel activity is controlled by the physiological range of pHi (Ki of pHi 7.35~7.48) (1, 2, 43-45). Astrocytes express a variety of transporters, including the cotransporter and Na+/H+ exchanger (46-48), both of which induce intracellular alkalinization when activated, and an increase in [K+]o during neural excitation causes up-regulation of cotransporter activity (49-51). This alkalinization would enhance the activity of heteromeric Kir4.1/5.1 channels (1, 2, 43-45) and facilitate the uptake of K+ by astrocytes at the synaptic site. On the other hand we found that the homomeric Kir4.1 dominated astrocyte processes in brain areas such as the stratum radiatum in the hippocampus, where little expression of cotransporter was detected (Ref. 52; see also Fig. 2).

Putative different roles for heteromeric Kir4.1/5.1 and homomeric Kir4.1 channels could also be seen in the olfactory bulb. Olfactory stimuli may evoke a larger increase in [K]o in intraglomerular regions where many synaptic connections are formed compared with inter-glomerular areas that contain a few synapses (53). The former would require more efficient K+ buffering than the latter, and we actually found that heteromeric Kir4.1/5.1 dominates at the inside of the glomeruli, and homomeric Kir4.1 is on the outside (Fig. 2). The K+-buffering function of Kir4.1/5.1 would then be boosted by its coupling with cotransporter since the transporter is expressed in astrocytes throughout the bulb (52, 54).

The Kir channels at the astrocyte processes facing the pia mater and blood vessels in the brain and those at end feet and perivascular processes of retinal Müller cells would both be responsible for extrusion of excess intracellular K+. This study showed that Kir4.1/5.1 heteromers are localized at the extrusion site in the brain astrocytes, which is different from retinal Müller cells, where Kir4.1-homoers are at the site (18). This suggests that functional control of K+ extrusion may differ between brain astrocytes and retinal Müller cells. Further studies are needed to clarify the physiological significance of this difference.

Syntrophins May Be Involved in the Targeting of Glial Kir Channels—The present study showed that, in brain astrocytes, heteromeric Kir4.1/5.1 channels locate predominantly at the perivascular processes, whereas Kir4.1/5.1 or homomeric Kir4.1 does so at the perisynaptic processes. Therefore, the divergent mechanisms may control the targeting of the homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels to specific membrane domains within an astrocyte.

An essential role of DAPC in targeting of homomeric Kir4.1 channels to end feet and perivascular processes in retinal Müller cells is indicated in the cultured cells (14, 55) and mdx3Cv mice, whose dystrophin gene was chemically disrupted (22). It is, however, of note that mdx3Cv mice remain to express Kir4.1 at the perisynaptic processes of Müller cells, where it assembles with Kir5.1. This suggests that unidentified mechanisms other than DAPC also participate in the control of targeting of Müller cell Kir channels. In the brain neocortex and hippocampus, on the other hand, DAPC was shown distributed at perivascular rather than perisynaptic processes of the astrocytes (56, 57), where we found Kir4.1/5.1-heteromer, but not Kir4.1 homomer, dominated (Fig. 2). We also show that some syntrophins can bind to Kir4.1 via its PDZ domain (Fig. 4) whether or not this formed monomeric or heteromeric channels with Kir5.1. These results suggest a possible role of DAPC proteins in scaffolding Kir4.1/5.1 channels in brain astrocytes as well. However, the mechanism is unclear for the apparently different roles of DAPC between brain astrocytes and retinal Müller cells in controlling heteromeric Kir4.1/5.1 versus homomeric Kir4.1.

The exact molecules of DAPC that directly associate with Kir4.1 or Kir4.1/5.1 remain unknown. Although biochemical binding assays revealed that a PDZ protein {alpha}-syntrophin could be an interaction partner for astrocyte Kir4.1 (23), it is noted that only some, but not all, of the {alpha}-syntrophin knockout mice ({alpha}-Syn-/-) exhibited modest defects in Kir4.1 expression (58). Thus, {alpha}-syntrophin may be at least partly involved in but not be exclusively responsible for the trafficking of Kir4.1-containing channels in the brain. Other factors and/or molecules such as {beta}1 and {beta}2 syntrophins that we found bind to the channels (Fig. 4) and also the constituents of DAPC other than syntrophins may be additionally or alternatively required for the process. Further studies are needed to elucidate the precise sorting mechanisms of the glial Kir channels.


   FOOTNOTES
 
* This work was supported by Grant-in-aid for Specific Research on Priority Area (2) 12144207 (to Y. K.), Grant-in-aid for Young Scientists 15790134 (to H. H.), and Japan-France Integrated Action Program (SAKURA) (to Y. K.) from the Ministry of Education, Science, Sports, and Culture of Japan, by Uehara Memorial Foundation (to Y. K.), by Kanae Foundation for Life and Socio-Medical Science (to H. H.), and by Inamori Foundation (to H. H.). 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

To whom correspondence should be addressed. Tel.: 81-6-6879-3512; Fax: 81-6-6879-3519; E-mail: ykurachi{at}pharma2.med.osaka-u.ac.jp.

1 The abbreviations used are: DAPC, dystrophin-associated protein complex; GST, glutathione S-transferase; GFP, green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Ian Findlay (Université de Tours, Tours, France) and Christoph Lossin (Osaka University, Osaka, Japan) for critical reading of this manuscript.



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 ABSTRACT
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 DISCUSSION
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
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