Expression of Kir4.1 and Kir5.1 inwardly rectifying potassium channels in oligodendrocytes, the myelinating cells of the CNS

The inwardly rectifying K+ channel subtype Kir5.1 is only functional as a heteromeric channel with Kir4.1. In the CNS, Kir4.1 is localised to astrocytes and is the molecular basis of their strongly negative membrane potential. Oligodendrocytes are the specialised myelinating glia of the CNS and their resting membrane potential provides the driving force for ion and water transport that is essential for myelination. However, little is known about the ion channel profile of mature myelinating oligodendrocytes. Here, we identify for the first time colocalization of Kir5.1 with Kir4.1 in oligodendrocytes in white matter. Immunolocalization with membrane-bound Na+/K+-ATPase and western blot of the plasma membrane fraction of the optic nerve, a typical CNS white matter tract containing axons and the oligodendrocytes that myelinate them, demonstrates that Kir4.1 and Kir5.1 are colocalized on oligodendrocyte cell membranes. Co-immunoprecipitation provides evidence that oligodendrocytes and astrocytes express a combination of homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels. Genetic knock-out and shRNA to ablate Kir4.1 indicates plasmalemmal expression of Kir5.1 in glia is largely dependent on Kir4.1 and the plasmalemmal anchoring protein PSD-95. The results demonstrate that, in addition to astrocytes, oligodendrocytes express both homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels. In astrocytes, these channels are essential to their key functions of K+ uptake and CO2/H+ chemosensation. We propose Kir4.1/Kir5.1 channels have equivalent functions in oligodendrocytes, maintaining myelin integrity in the face of large ionic shifts associated with action potential propagation along myelinated axons.


Introduction
Glial cell function is largely determined by their expression of a wide range of plasmalemmal potassium channels (Verkhratsky and Steinhäuser 2000). The inwardly rectifying potassium channel subtype Kir4.1 is the dominant K ? channel in astrocytes (Kofuji and Newman 2004). Studies using the global Kir4.1 knock-out and targeted ablation have demonstrated the critical role for Kir4.1 in the key astroglial function of potassium homeostasis (Kofuji et al. 2000;Neusch et al. 2006;Djukic et al. 2007;Seifert et al. 2009;Bay and Butt 2013). In addition, astrocytes express multiple Kir subtypes and their functional diversity is increased by the formation of both homomeric and heteromeric channels (Hibino et al. 2010;Butt and Kalsi 2006). Kir5.1 is an inwardly rectifying K ? channel subunit that forms functional heteromeric channels with Kir4.1 (Konstas et al. 2003). Heteromeric Kir4.1/5.1 channels are highly pH sensitive and have biophysical properties distinct from homomeric Kir4.1 channels (Tanemoto et al. 2000;Pessia et al. 2001). Heteromeric Kir4.1/Kir5.1 channels have been indicated in astrocytes of the retrotrapezoid nucleus (RTN), where due to their special pH/CO 2 sensitivity they are important in chemoreception and contribute to respiratory drive (Mulkey and Wenker 2011). The relationship between expression of Kir5.1 with the dominant astrocyte channel Kir4.1 is not fully elucidated, but it is possible that homomeric Kir4.1 and heteromeric Kir5.1/Kir4.1 channels may be differentially expressed in specific astroglial populations (Ishii et al. 2003;Hibino et al. 2004).
The other main class of glia in the CNS are oligodendrocytes, which have the essential function of myelinating axons that make up CNS white matter. In contrast to astrocytes, the ion channel profile of mature myelinating oligodendrocytes is unresolved. There is immunohistochemical evidence that oligodendrocytes express Kir4.1 (Poopalasundaram et al. 2000;Kalsi et al. 2004), and a study in the global Kir4.1 knock-out mouse demonstrates it is essential for oligodendrocyte development and myelination (Neusch et al. 2001). However, oligodendroglial expression of Kir4.1 is controversial (Hibino et al. 2004;Tang et al. 2009), and Kir5.1 has not been studied previously in an oligodendroglial context. Here, we have determined the expression of Kir5.1 and Kir4.1 in oligodendrocytes in the white matter of the mouse cerebellum and optic nerve, in comparison to astrocytes. The optic nerve is a typical CNS white matter tract that contains the axons of retinal ganglion cells and the glia that support them and is ideal for examining myelinating oligodendrocytes ). The results demonstrate that oligodendrocytes, like astrocytes, express at least two subsets of Kir channels, homomeric Kir4.1 channels and heteromeric Kir4.1/5.1 channels. Oligodendroglial K ? channels are essential for myelin formation and integrity in the face of depolarizing elements (Hawkins and Butt 2013;Neusch et al. 2001;Menichella et al. 2006). The special biophysical properties of homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels indicate they have distinct physiological roles in maintaining oligodendrocyte function during the large shifts in K ? and pH to which they are exposed during action potential propagation.

Experimental animals
All animals were killed by cervical dislocation, in accordance with regulations issued by the Home Office of the United Kingdom under the Animals (Scientific Procedures) Act, 1986. The animals used were C57BL6/J wild type strains and GFAP-EGFP, PLP-DsRed, Sox10-EGFP and Kir4.1 knock out transgenic mouse strains: GFAP-EGFP mice express enhanced green fluorescent protein (EGFP) under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter (Nolte et al. 2001); PLP-DsRed mice express DsRed under the control of the oligodendrocyte-specific proteolipid protein (PLP) promoter (Hirrlinger et al. 2005); Sox10-EGFP mice express EGFP under the control of the Sox10 promoter, which in the CNS is specific to oligodendrocyte lineage cells (Stolt et al. 2006); the GFAP-EGFP and PLP-DsRed strains were kindly provided by Frank Kirchhoff (Molecular Physiology, University of Saarland, Homburg, Germany) and the Sox10-EGFP by Bill Richardson (Wolfson Institute for Biomedical Research, University College London, UK). The K ir 4.1 channel knock-out mice were originally generated by Kofuji et al. (2000), and were kindly provided by Christian Steinhäuser (Institute of Cellular Neurosciences, University of Bonn, Germany). Kir4.1 KO mice were kept in a heterozygous background and genotyping was performed to identify homozygous, heterozygous and wild type offspring using the Mouse Tail Quick Extraction Solutions (Biopioneer Inc) and PCR amplification, as previously described (Kofuji et al. 2000).

Optic nerve explant cultures
Optic nerve explant cultures were prepared from mice aged postnatal day (P)7-11, as described previously (Hawkins and Butt 2013). In brief, optic nerves were carefully dissected and maintained in pre-warmed (37°C) and pregassed (95 % O 2 /5 % CO 2 ) dissecting media, constituted of Dulbecco's Modified Eagle Medium (DMEM) (Sigma) medium, supplemented with 4 mM L-glutamate, 10 % foetal bovine serum (FBS; Invitrogen) and 0.1 % gentamicin (Invitrogen, Life Technologies Ltd., Paisley). From this point on optic nerves were kept under sterile conditions and cut into 1-2 mm fragments in filter sterilized prewarmed dissecting media, using a scalpel blade. For further dissociation, optic nerve fragments were triturated and nerve fragments transferred onto laminin (Invitrogen) and poly-l-ysine (Sigma) coated coverslips and incubated in dissecting medium at 37°C in 95 % O 2 /5 % CO 2 overnight to allow the adhesion of the explants. After 24 h, the dissecting media was substituted with a low serum (0.5 %) modified Bottenstein and Sato (B&S) culture medium, supplemented with 10 ng/ml recombinant human PDGF-AA (R&D Systems) and 0.1 % gentamicin. After 3-4 days in vitro (DIV) the medium was replaced to B&S media supplemented with 0.5 mM dibutyryl cAMP for up to 10 DIV. Explanted cells were then cultured in B&S media with 0.1 % gentamicin for up to 15 DIV, changing media every 3-5 days.

Image capture and analysis
Fluorescence was detected using excitation wavelengths of 488 nm (green), 568 nm (red), 633 nm (far red) and 405 nm (blue), with an argon, HeNe1 and diode laser, respectively. Confocal images were captured using the 409 or 639 oil immersion objectives (numerical apertures of 1.3 and 1.4, respectively), and images acquired using multi-track sequential capture, with optimal detector gain and offset acquisition settings for pinhole diameter 0.13-0.3 airy units, with an average of four scans per image, to detect positive signal with minimal background and prevent cross-talk between channels. Identical settings were used to image negative controls. Z-stacks were captured of 4-15 z-sections (voxel size 43-76 nm x-y, 76-283 nm z). Image analysis was carried out using Volocity 6.1 software (Improvision Ltd.). For confocal photomicrographs, two-dimensional flattened images of the z-stacks are presented. For colocalization analyses, the technique of Barlow and colleagues was used (Barlow et al. 2010), as previously described (Hawkins and Butt 2013), in which the degree of separation between pixels from the red and green channels was determined in single z-sections to provide measurements of signal overlap. First, images were thresholded to separate the positive signal (positive immunolabelling) from background; threshold was determined by measuring the background intensity value for each channel in negative control sections and setting the threshold as the mean background intensity plus three standard deviations (averaged from a minimum of 6 images). The thresholded Pearson's correlation coefficient (PCC) and Mandersons' overlap coefficients (M1 and M2) were determined as previously described (Barlow et al. 2010), using Volocity 6.1 software; thresholded PCC determines the statistical strength of the linear relationship between fluorescent intensities from the red and green channels, and the M1 and M2 overlap coefficients provide accurate measurements of the true degree of overlap of red and green. A colocalization channel was generated from the thresholded PCC to illustrate in three-dimensions the voxels in which the two channels overlap with the same intensity.

Isolation of plasma membrane fraction
Whole brains and optic nerves of wild type and Kir4.1 KO animals were homogenised in subcellular fractionation buffer containing 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 DTT and 19 protease inhibitor cocktail. Samples were centrifuged at 4°C with 750 G for 10 min to remove the nuclear fraction. Supernatant was placed in a new Eppendorf tube and centrifuged with 10,000g at 4°C two times to remove the mitochondrial fraction. Supernatant was placed in an ultracentrifuge tube and centrifuged with 40,000g at 4°C for 1 h. The supernatant was removed and the pellet containing the crude plasma membrane fraction was resuspended in 400 ll fractionation buffer using a 25 G needle and centrifuged at 40,000g at 4°C for 45 min and then the pellet was resuspended in lysis buffer.

Immunoprecipation
Brains and optic nerve obtained from adult wild type mice (P16-40) were homogenised as described above. For the precipitation of the proteins lMACS TM Protein A/G MicroBead kit was used (Miltenyi Biotec) according to the manufacturers recommendations. In brief, homogenised brain or optic nerve samples were centrifuged at 4°C with 10,000g 2 times to clean the homogenate from cell debris. 3 lg polyclonal-or 2 lg monoclonal antibody was added to the proteins and incubated overnight at 4°C. In the case of negative controls, proteins were mixed with the preabsorbed antibodies or for Kir4.1, Kir4.1 knock out tissue was used. Then, Protein G MicroBeads were added to the samples to magnetically label the immune complex (50 ll if monoclonal antibody was used or 100 ll if polyclonal antibody was used for the precipation). The samples were then mixed and incubated for 30 min on ice. The new l Column were placed in the magnetic field of the lMACS TM separator and then prepared by rinsing with 200 ll of lysis buffer containing of 150 mM NaCl, 1 % Triton X-100, 50 mM Tris HCl (pH 8.0), before the cell homogenate was applied onto the column. The homogenate run through the columns was washed with 200 ll lysis buffer four times then with 100 ll of low-salt wash buffer containing of 1 % NP-40, 50 mM Tris HCl (pH 8.0). To prepare the elution of the immune complex 20 ll of preheated (95°C) 1 9 Laemelli sample buffer was applied onto the column and incubated for 5 min at RT. Next, 50 ll of pre-heated (95°C) 19 Laemmli sample buffer was applied to the column matrix to elute the immune complex. The drop on the column tip containing the eluted immunoprecipitate was collected and stored on ice or at -80°C until it was analyzed by SDS-PAGE.

Results
Oligodendrocytes and astrocytes express Kir4.1 and Kir5.1 in the brain The Kir5.1 subunit forms functional channels as heteromers with Kir4.1, hence Kir5.1 expression might be expected to mirror that of Kir4.1 in the brain (Hibino et al. 2004). Kir4.1 is established as the main Kir subunit in astrocytes and is highly expressed in the cerebellum (Tang et al. 2009). In contrast, oligodendroglial expression of Kir4.1 is poorly defined, but they are reported to be immunopositive in the cerebellum (Poopalasundaram et al. 2000). Therefore, we assessed the expression of Kir5.1 in comparison with Kir4.1 in cerebellar astrocytes and oligodendrocytes (Fig. 1). Double immunofluorescence labeling demonstrated extensive colocalization between GFAP and Kir4.1, with strong and distinctive immunopositivity for Kir4.1 in the radial processes of Bergmann glia extending from the Purkinje cell layer through the molecular layer to the pia, together with astrocyte somata and processes in the granule cell layer and white matter (Fig. 1A). In contrast, the radial processes of Bergmann glia were not delineated by Kir5.1 immunolabelling, and Kir5.1 was most prominent in the granule cell layer and included distinct co-localisation with GFAP in astrocytes (Fig. 1C). The results indicate differential expression of Kir4.1 and Kir5.1 in Bergmann glia and cerebellar astrocytes. Expression of Kir4.1 and Kir5.1 in oligodendrocytes was examined using the APC(CC1) antibody (Bhat et al. 1996), Olig2 immunolabelling, and in sections from mice that express EGFP under the control of the oligodendrocyte-specific Sox10 promoter (Stolt et al. 2006); APC, Olig2 and Sox10 are essential for oligodendrocyte differentiation and their expression is routinely and extensively used to identify oligodendrocytes (Bhat et al. 1996;Bay and Butt 2013;Lang et al. 2013;Azim et al. 2014;Fancy et al. 2014). Although neurons also express APC, it does not appear to be recognized by the APC/CC1 antibody and may be a different isoform than found in oligodendrocytes (Brakeman et al. 1999). Double immunofluorescence labeling with APC(CC1) to label oligodendrocyte somata (Fig. 1B, D) and immunolabelling in Sox10-EGFP? cells (Fig. 1E, F) demonstrates localization of Kir4.1 and Kir5.1 in oligodendrocytes of the cerebellar white matter. Further evidence of oligodendroglial expression of Kir4.1 is provided by immunolabelling with Olig2 (Fig. 1G). The results demonstrate robust expression of Kir4.1 and Kir5.1 in oligodendrocytes in the cerebellar white matter. No immunoreactivity was detected in negative controls in sections from knock out mouse for Kir4.1 (Fig. 1Aiv) and by preabsorbing the primary antibody with antigen peptide for Kir5.1 (Fig. 1-Civ). In addition, the specificity of the antibodies was further confirmed by western blot analysis of total protein lysates in the brain and optic nerve (Fig. 1I, J). For Kir4.1, the predicted molecular weights of the monomeric 42 kDa, heteromeric 80 kDa, and tetrameric 160 kDa channels were identified in wild-type mice, and these were absent in brain lysates from Kir4.1 knock-out mice (Fig. 1I). Western blot for Kir5.1 was performed in wild type mice, with a predicted molecular weight of 48 kDa, together with bands at 36 kDa and 70 kDa, depending on the extent of glycosylation of the cytoplasmic and membrane bound channels (Hibino et al. 2004), and positive bands were eliminated by the competitive peptide (Fig. 1J).

Localization of Kir4.1 and Kir5.1 in optic nerve astrocytes and oligodendrocytes
The optic nerve is a typical myelinated CNS white tract and immunolocalization of Kir4.1 has been clearly identified in astrocytes and oligodendrocytes (Kalsi et al. 2004). We therefore examined the relationships between Kir5.1 and Kir4.1 in optic nerve glia, using transgenic reporter mice in which EGFP is under the control of the astrocyte-specific promoter for the human GFAP gene and DsRed is under the control of the oligodendrocyte-specific PLP gene (Fig. 2). Extensive immunostaining for Kir4.1 and Kir5.1 was localised both in transversely oriented astrocytes ( Fig. 2A, B) and longitudinal rows of oligodendrocytes (Fig. 2C, D). Colocalization of fluorescence labelling is most frequently presented as overlays of red and green channels, with areas of yellow indicating colocalization (Fig. 2Aiv, Biv, Civ, Div). To analyse this more accurately, we used the technique of Barlow and colleagues (Barlow et al. 2010), to perform quantification of the degree of colocalization between Kir4.1 and Kir5.1 with GFAP-EGFP and PLP-DsRed on high resolution confocal z-sections (Bay and Butt 2013). In brief, the background intensity measured in negative control sections (averaged from a minimum of 6 images) was used to threshold the images and determine the Pearson's correlation coefficients in single z-sections, from which colocalization channels were generated. In this way, the colocalization channel identifies the individual voxels in which the two channels overlap with the same intensity (Fig. 2Av,Bv,Cv,Dv). The results demonstrate optic nerve astrocytes and oligodendrocytes express Kir5.1 and Kir4.1 throughout their cell somata and processes.
Plasmalemmal expression of Kir4.1 and Kir5.1 subunits in optic nerve glia To be functional, Kir4.1 and Kir5.1 subunits need to be expressed in the plasmalemma and this was assessed by two methods: immunocolocalization with the plasma membrane marker Na ? -K ? -ATPase a1 subunit in optic nerve glial explant cultures, and western blot analysis of purified plasma membrane fractions (Fig. 3). Immunolabelling of optic nerve explant cultures demonstrates Kir4.1 and Kir5.1 colocalise with Na ? -K ? -ATPase in both GFAP-positive astrocytes (Fig. 3A, B) and PLP-positive oligodendrocytes (Fig. 3C, D). Quantification of co-localization, as described above, demonstrated significant colocalisation of both Kir4.1 and Kir5.1 with Na ? -K ? -ATPase in astrocytes and oligodendrocytes (Fig. 3E, F). Western blot analysis of plasmamembrane fractions compared to total lysates confirmed plasmalemmal expression of Kir5.1 and Kir4.1 subunits in the optic nerve (Fig. 3G,  H). Measurement of the density of the bands relative to bactin indicated a 3.9-fold enrichment of Kir5.1 in the optic nerve plasma membrane fraction, compared to 2.1-fold enrichment for Kir4.1; no bands were observed in the negative controls, using the Kir4.1 knock-out mouse and pre-incubation in the Kir5.1 blocking peptide. In addition, PSD-95 clusters Kir channels in the cell membrane through its interaction with their PDZ domains (Horio et al. 1997;Tanemoto et al. 2002;Pegan et al. 2007), hence we used co-immunprecipitation of total brain and optic nerve lysates to determine whether PSD-has a role in anchoring Kir4.1 and Kir5.1 in optic nerve glia (Fig. 3I, J). PSD-95 was co-immunoprecipitated with both Kir4.1 (Fig. 3I) and Kir5.1 (Fig. 3J) in the brain and optic nerve; western blot of the anti-PSD-95 antibody gave a dense band at the b Fig. 1 Expression of Kir4.1 and Kir5.1 in oligodendrocytes and astrocytes in the cerebellum. Immunolabelling for Kir4.1 and Kir5.1, in combination with GFAP for astrocytes (A, C), and APC/CC1 for oligodendrocytes (B, D). Immunolabelling for Kir4.1 (E) and Kir5.1 (F) in mice in which EGFP is under the control of the oligodendrocyte-specific Sox10 promoter. G Double immunolabelling for Kir4.1 (red) and the oligodenrocyte-specific marker Olig2 (green). Insets in Aiv and Civ illustrate negative controls, in the Kir4.1 KO mouse (Aiv) and following preincubation with the Kir5.1 blocking peptide (Civ). Scale bars 20 lm. Western blot analysis of the brain and optic and nerve for Kir4.1 (I) and Kir5.1 (J); bands were absent in the negative controls, in the Kir4.1 knock-out mouse (I) following preincubation in the Kir5.1 blocking peptide (J) .1 is demonstrated by the generation of colocalisation channels (Av, Bv, Cv, Dv) from confocal z-stacks (Aiv, Biv, Civ, Div), and green and red channels of equal intensity appear yellow. Scale bars 20 lm predicted MW and no band was detected in the negative controls, and no co-immunoprecipitiation with PSD-95 was observed in the negative controls, using the knock-out mouse for Kir4.1 (Fig. 3I) and blocking peptide for Kir5.1 (Fig. 3J). These results demonstrate Kir4.1 and Kir5.1 subunit expression in the glial plasmalemma and support a role for PSD-95 in their membrane localization.
Similarly, western blot of the brain plasma membrane fraction from wild-type and Kir4.1 knock-out mice demonstrated a significant reduction in Kir5.1 levels by 60 ± 14 % in the absence of Kir4.1 (Fig. 6D, E). These results demonstrate an overall decrease in Kir5.1 expression in the absence of Kir4.1 and a specific reduction in plasmalemmal Kir5.1, providing evidence that the bulk of glial Kir5.1 is expressed in the form of heteromeric b Fig. 3 Plasmalemmal expression of Kir4.1 and Kir5.1 subunit in optic nerve glia. Immunolocalization of Kir4.1 and Kir5.1 with the membrane bound Na-K-ATPase a1 subunit in optic nerve explants of astrocytes identified by GFAP (A, B) and oligodendrocytes identified by PLP-DsRed (C, D). Scale bars 20 lm. Quantification in astrocytes and oligodendrocytes of total number of voxels immunopositive for Kir4.1 and Kir5.1, compared to voxels that were identified as colocalized for Kir4.1/Na-K-ATPase (E) and Kir5.1/Na-K-ATPase (F); data are mean ± SEM, n = 13 cells for each analysis. Western blot analysis of Kir5.1 (G) and Kir4.1 (H) in total optic nerve lysate and plasma membrane fraction. Co-immunoprecipitation of Kir4.1 (I) and Kir5.1 (J) with PSD95, in total brain and optic nerve (ON) lysate; negative controls were Kir4.1 knock-out mice (-/-) for Kir4.1 and preincubation with the blocking peptide for Kir5.1 channels with Kir4.1, and most of the remaining Kir5.1 subunits appear to be retained intracellulary and do not form plasmalemmal channels in the absence of the Kir4.1.
Kir5.1 is decreased in myelin in the absence of Kir4.1 Oligodendrocytes were very rarely transfected in our in vitro experiments and so we examined interactions between Kir5.1 and Kir4.1 in oligodendrocytes by immunolabelling of optic nerve and brain sections from Kir4.1 knock-out mouse. There is a complete loss of Kir4.1 immunolabeling in the knock-out mouse (Fig. 1Aiv), and this results in an evident decrease in Kir5.1 immunolabelling overall, as well as a specific decrease in Kir5.1 in oligodendrocytes and myelin, as illustrated by MBP immunolabelling in the optic nerve (Fig. 7A, B), and by APC(CC1) immunolabeling in the corpus callosum (Fig. 7C, D) and cerebellum (Fig. 7E, F). We showed above by western blot a complete loss of Kir4.1 protein in the knock-out mouse (Fig. 1I), and this resulted in significant decreases in Kir5.1 protein levels in the optic nerve ( Fig. 7G) and brain (Fig. 7H), by 33.5 ± 3.5 and 53 ± 0.4 %, respectively ( Fig. 7I; p \ 0.001, unrelated t test).
Oligodendrocytes and white matter astrocytes express Kir4.1 and Kir4.1/5.1 Co-immunoprecipitation and immunolabeling identified the existence of both homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels in astrocytes and oligodendrocytes of optic nerve and cerebellar white matter. The anti-Kir4.1 antibodies used in this study have been validated previously (Kalsi et al. 2004) and their specificity was confirmed using western blot and immunohistochemistry in Kir4.1 knock-out mice, in support of previous studies using a similar antibody (Kofuji et al. 2000;Neusch et al. 2001). In the case of Kir5.1, the specificity of the antibodies was confirmed by using blocking peptides and comparing two commercially available antibodies from Alomone (rabbit anti-rat Kir5.1 antibody) and Santa Cruz (goat anti-mouse Kir5.1 antibody), raised againist two different non overlapping epitopes in the C-terminus of the channel. The two antibodies provided equivalent immunoblot and immunolabeling results that were completely absent in the presence of blocking peptide. Expression of Kir4.1 and Kir5.1 in oligodendrocytes was verified in three ways: (1) in double immunolabelling with the anti-APC(CC1) and Olig2 antibodies-APC and Olig2 are essential for oligodenrocyte differentiation and the CC1 antibody is used routinely and extensively to identify oligodendrocytes in the CNS (Bhat et al. 1996;Bay and Butt 2013;Lang et al. 2013;Azim et al. 2014;Fancy et al. 2014); (2) immunolabelling in sections from mice that express EGFP under the control of the Sox10 promoter, which is a transcription factor that is essential for oligodendrocyte differentiation and is expressed by all oligodendrocyte lineage cells (Stolt et al. 2006;Azim et al. 2014); (3) immunolabelling in sections and isolated cells from mice that express DsRed under the control of the myelin protein PLP (Hirrlinger et al. 2005;Azim et al. 2014). The results demonstrate expression of Kir4.1 and Kir5.1 in astrocytes and oligodendrocytes, and specifically plasmalemmal localisation of the Kir subunits on glia by immunoblot of optic nerve plasma membrane and immunocytochemical co-localisation with plasma b Fig. 4 Co-expression of Kir4.1 and Kir5.1 in optic nerve oligodendrocytes and astrocytes. Co-immunolocalization of Kir4.1 and Kir5.1 in optic nerve explant cultures, in astrocytes identified by GFAP immunolabelling (A) and oligodendrocytes identified by PLP-DsRED (B). The overlay and individual channels are illustrated, together with the co-localisation channel for Kir4.1/Kir5.1 (Aii, Bii). Boxed areas on overlay images (Ai, Bi) are enlarged in Avi-Aviii and Bvi-Bviii, to illustrate punctate colocalization of Kir4.1 and Kir5.1 along processes (some indicated by arrows). Scale bars 20 lm. Quantification of the number of voxels that were positive for Kir4.1 and Kir5.1 alone and of Kir4.1/Kir5.1 together, in astrocytes (C, n = 15) and oligodendrocytes (D, n = 13); data are mean ± SEM. Co-immunoprecipitation of Kir4.1 with Kir5.1 (E) and of Kir5.1 with Kir4.1 (F) from total brain and optic nerve (ON) lysates; negative controls were Kir4.1 knock-out mice (-/-) for Kir4.1, and using the blocking peptide for Kir5.1 membrane-bound Na-K-ATPase. Immunoblot analysis demonstrated robust bands as predicted according to the momomer subunit molecular weights, 42 kDa for Kir4.1 and 48 kDa for Kir5.1. In addition, bands for Kir4.1 were indicated at 80 and 160 kDa, which corresponds to the heteromeric and homo-tetrameric channel as observed previously in the brain and retina (Kofuji et al. 2000;Ulbricht et al. 2008). Furthermore, bands were detected at 70 kDa for Kir5.1, which were more enriched in the optic nerve plasma membrane fraction, and are consistent with previous findings indicating the ion channel is regulated by glycosylation (Ishii et al. 2003). A key finding was that the co-immunoprecipitation and immunocytochemical analyses indicated oligodendrocytes and astrocytes express Kir4.1/Kir5.1 heteromers, with the majority of Kir5.1 being associated with Kir4.1 and present in the plasmalemma, which is essential for functional heteromeric channels. Furthermore, examination of Kir4.1 knock-out mouse tissue and using shRNA to ablate Kir4.1 in vitro demonstrated that plasmalemmal Kir5.1 expression was almost completely dependent on Kir4.1. In contrast, the bulk of Kir4.1 ([80 %) in optic nerve glia was not co-expressed with Kir5.1, indicating a predominance of homomeric Kir4.1 channels.
Heterogeneity of glial Kir4.1 and Kir5.1 expression Astroglial Kir4.1 and Kir5.1 immunolabelling was heterogeneous in the brain and optic nerve, indicating that most astrocytes expressed Kir4.1 and Kir5.1 to a high degree, but a subpopulation were only weakly immunopositive or possible immunonegative for Kir5.1, in agreement with previous studies (Poopalasundaram et al. 2000;Hibino et al. 2004;Kalsi et al. 2004;Tang et al. 2009). In the cerebellum, Bergmann astroglia were distinctive in that their primary processes were strongly decorated with Kir4.1, but not Kir5.1, whereas astrocytes in the granule cell layer expressed Kir4.1 and Kir5.1. White matter astrocytes were enriched with Kir4.1, which is consistent with studies in knock-out mice demonstrating Kir4.1 are essential for prominent astroglial inward currents and the uptake of K ? released by axons during action potential propagation (Kofuji et al. 2000;Neusch et al. 2006;Djukic et al. 2007;Seifert et al. 2009;Bay and Butt 2013). A prominent role for heteromeric Kir4.1/Kir5.1 channels in K ? transport has also been demonstrated in transporting epithelia, for example in the renal tubules and gastric parietal cells (Paulais et al. 2011;Tanemoto et al. 2000;Tucker et al. 2000;Lachheb et al. 2008). Heteromeric Kir4.1/Kir5.1 channels are stronger inward rectifiers than homomeric Kir4.1 channels, which provides a potential mechanism for the uptake of K ? at sites of high activity via Kir4.1/Kir5.1 and release of K ? at sites of low activity via homomeric Kir4.1 channels (Kofuji et al. 2002;Butt and Kalsi 2006;Bay and Butt 2013). The close association of Kir4.1 and Kir5.1 with plasmalemmal Na ? -K ? -ATPase suggests the targeting of these channels to the same membrane microdomains may help provide K ? for the activity of the pumps, as observed for H ? -K ? -pumps in parietal cells Kaufhold et al. 2008).
In the CNS, heterometric Kir4.1/Kir5.1 channels have been described in retinal Müller cells, brain astrocytes derived from neocortex, retrotrapezoid nucleus and the glomeruli of the olfactory bulb, and brainstem neurons from the cardio-respiratory nuclei (Hibino et al. 2004;Ishii et al. 2003;Tanemoto et al. 2000;Tucker et al. 2000;Wu et al. 2004;Yamamoto et al. 2008). Interestingly, a study using a bacterial artificial chromosome (BAC) transgenic approach to express EGFP under the transcriptional control of the Kir4.1 promoter reported prominent expression of EGFP in astrocytes but not oligodendrocytes (Tang et al. 2009). In contrast, Kir4.1 have been detected in oligodendrocytes by immunohistochemistry (Poopalasundaram et al. 2000;Kalsi et al. 2004) and electrophysiological experiments demonstrate prominent Kir in oligodendrocytes (Neusch et al. 2001;, and genetic ablation of Kir4.1 results in a loss of Kir currents and causes severe dysmyelination (Neusch et al. 2001). Our results demonstrate that in white matter of the optic nerve and cerebellum oligodendrocytes express heteromeric Kir4.1/Kir5.1 channels, as well as homomeric Kir4.1 channels, and we show the loss of Kir4.1 is associated with a marked reduction of Kir5.1 in oligodendrocytes and myelin, supporting a role for these channels in oligodendroglial K ? regulation and myelin integrity (Neusch et al. 2001;Menichella et al. 2006). The discrepancy in the literature is likely to reflect the heterogeneity of oligodendroglial expression of Kir4.1, which appears greatest in the optic nerve, cerebellum and spinal cord, whereas in the forebrain Kir4.1 is far more prominent in protoplasmic astrocytes compared to oligodendrocytes (Poopalasundaram et al. 2000;Neusch et al. 2001;Kalsi et al. 2004).  (Konstas et al. 2003;Tanemoto et al. 2004). In addition, we provide evidence that clustering of Kir4.1 and Kir5.1 involves the MAGUK family PDZ-binding anchoring ; transfected cells were identified by cotransfection with GFP (appears green) and the co-localization channel indicates voxels in which Kir5.1 and Na-K-ATPase immunolabelling was at the same intensity (Avi, Bvi). Scale bars 20 lm. C Quantification of plasmalemmal Kir5.1 expressed as percentage of total Kir5.1 ? voxels (data are mean ± SEM, n = 11-13 per group; *p \ 0.05, one-tailed t test). D, E Western blot of Kir5.1 expression in the brain plasma membrane fraction from Kir4.1 ?/? wild-type and Kir4.1 -/knock-out mice (D) and mean (±SEM) integrated density normalised against b-actin (E, n = 3, ***p \ 0.001, one-tailed t test) proteins PSD-95, which has been described in other cells (Horio et al. 1997;Tanemoto et al. 2002). Although Kir5.1 homomeric channels are generally thought to be nonfunctional, there is evidence that clustering of Kir5.1 subunits by PSD-95 mediates the formation of functional homomeric Kir5.1 channels in the brain (Tanemoto et al. 2002). We observed co-immunoprecipitation of Kir5.1 with PSD-95 in the optic nerve and a high degree of Kir5.1 immunolabelling in optic nerve glia was not associated with Kir4.1, raising the possibility they may express functional homomeric Kir5.1 channels. However, Kir5.1 may also form heteromers with other Kir subtypes, including Kir2.1 (Derst et al. 2001;Pessia et al. 2001), which is expressed by astrocytes (Horio 2001;Howe et al. 2008;Kang et al. 2008), and also clusters with PSD-95 (Fomina et al. 2011;Nehring et al. 2000;Leonoudakis et al. 2001;Pegan et al. 2007). Our results identified that Kir5.1 immunolabeling was mostly concentrated within the cell cytoplasm, tenfold greater than at the plasmalemma, indicating they provide a pool by which plasmallemal Kir4.1/Kir5.1 channels could be inserted into the cell membrane and provide dynamic regulation of the glial membrane potential in response to changes in the extracellular environment ). Kir5.1 are known to display intracellular localization and phosphorylation dependent interactions result in the targeting of Kir5.1/Kir4.1 heteromeric channels to the membrane (Tanemoto et al. 2000(Tanemoto et al. , 2004(Tanemoto et al. , 2008. Furthermore, Kir4.1/Kir5.1 channels are regulated by multiple factors, including PKA, PIP2, pH and CO 2 Yang et al. 2000;Rapedius et al. 2007;Rojas et al. 2007Rojas et al. , 2008. Hence, the cytoplasmic pool of Kir5.1 would allow astrocytes and oligodendrocytes to respond rapidly to changes in the extracellular environment and dynamically target heteromeric Kir4.1/Kir5.1 channels to the cell membrane to provide channels with much stronger rectification and pH/CO 2 sensitivity than Kir4.1 homomeric channels (Tanemoto et al. 2000;Yang et al. 2000;Pessia et al. 2001).
Functional implications of heteromeric Kir4.1/ Kir5.1 channels in oligodendrocytes Kir5.1 co-assembles with Kir4.1 to form functional pHsensitive potassium channels Cui et al. 2001;Pessia et al. 2001), which have been identified as Oligodendroglial expression of Kir4.1 channels indicates they may be important in uptake of excess K ? released during axonal action potential propagation, a function largely attribiuted to astrocytes. Due to their wrapping of axons, oligodendrocytes are exposed to large ionic and pH shifts during axonal electrical activity, and it is likely weakly rectifying homomeric Kir4.1 and strongly rectifying Kir4.1/Kir5.1 heteromeric channels are important in maintaining the negative resting membrane potential, which is essential for oligodendroglial and myelin integrity. Weakly rectifying homomeric Kir4.1 channels may preferentially extrude K ? and supply extracellular K ? for the Na ? -K ? -pumps, as described in transporting epithelia. In contrast, the pH sensitivity of heteromeric Kir4.1/Kir5.1 channels is likely to have a role in the CO 2 / pH chemosensation in glia, involving carbonic anhydrase that is enriched in astrocytes and oligodendrocytes. Furthermore, intracellular acidification and inhibition of Kir4.1/Kir5.1 channels has been shown to trigger release of ATP from astrocytes, which would act on oligodendroglial P2X and P2Y receptors to provide a mechanism of astrocyte-oligodendrocyte signaling in response to metabolic challenges, which has important implications for white matter physiology and pathology astroglial CO 2 chemoreceptors in the chemosensitive nuclei of the brainstem (Mulkey and Wenker 2011;Wu et al. 2004;Wenker et al. 2012). In white matter, oligodendrocytes are exposed to considerable ionic and pH shifts that take place during intense and continuous action potential propagation along axons (Kettenmann et al. 1990;Ransom 1992). This places a high metabolic load on oligodendrocytes and it is likely that Kir4.1/Kir5.1 channels in oligodendrocytes are involved in sensing changes in pH/CO 2 , which could be important in maintaining their RMP during metabolic stress (Fig. 8). As in other cells, a primary role of weakly rectifying homomeric Kir4.1 channels would be to supply Na ? -K ? pumps, which is essential for K ? regulation by astrocytes and oligodendrocytes, whilst heteromeric Kir4.1/Kir5.1 channels would be essential for pH/CO 2 sensation due to their unique pH sensitivity in the physiological range (Cui et al. 2001). Astrocytes express carbonic anhydrase (CA) V, while oligodendrocytes have CAII, which is critical for astroglial and oligodendroglial H ? regulation by the rapid conversion of CO 2 and H 2 O to H 2 CO 3 , which dissociates to HCO 3 and H ? coupled to plasmalemmal Na ? -H ? -exchange and Na ? -HCO 3 cotransport, as well as Na ? -independent Cl --HCO 3 --exchange (Kettenmann et al. 1990;Boussouf and Gaillard 2000;Ro and Carson 2004). Kir4.1/Kir5.1 currents are regulated by CO 2 and intracellular pH Cui et al. 2001), hence inhibition of Kir4.1/Kir5.1 channels in response to intracellular acidification during intense axonal electrical activity and high metabolic demand would reduce inward K ? currents and help maintain the oligodendroglial membrane potential, which is essential for myelin integrity (Hawkins and Butt 2013). Furthermore, in the retrotrapezoid nucleus of the central chemoreceptors, inhibition of astroglial Kir4.1/Kir5.1 currents is the mechanism by which they sense an increase in extracellular CO 2 and this triggers the release of ATP from astrocytes (Wenker et al. 2012). We have previously demonstrated that optic nerve astrocytes release ATP in response to axonal electrical activity (Hamilton et al. 2008(Hamilton et al. , 2010 and astroglial ATP stimulates local blood flow (Butt 2011). Thus, astroglial Kir4.1/Kir5.1 channels provide a mechanism by which they sense increased H ? /CO 2 during axonal electrical activity and release ATP to increase local blood flow and maintain axonal function. In addition, ATP released by astrocytes would directly act on metabotropic ATP receptors (purinoceptors) on oligodendrocytes, namely P2Y 1 and P2Y 12 subtypes, which are prosurvival and maintain myelin integrity ). However, in hypoxia/ischemia excessive release of ATP by astrocytes would activate oligodendroglial P2X 7 receptors, which would cause myelin destruction (Matute et al. 2007), and blockade of Kir4.1/Kir5.1 may be a therapeutic target under these pathological conditions.