Abstract
Inward Rectifying Potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis and neuronal excitability. The most recently described Kir subtype is Kir7.1, which is known as a K+ transporting subtype. Earlier studies localised Kir7.1 to subpopulations of neurones in the brain. However, the pattern of Kir7.1 expression across the brain has not previously been examined. Here, we have determined neuronal and glial expression of Kir7.1 in the adult mouse brain, using immunohistochemistry and transgenic mouse lines expressing reporters specific for astrocytes [glial fibrillary acidic protein‐enhanced green fluorescent protein (GFAP‐EGFP], myelinating oligodendrocytes (PLP‐DsRed), oligodendrocyte progenitor cells (OPC, Pdgfra‐cre ERT 2/Rosa26‐YFP double‐transgenic mice) and all oligodendrocyte lineage cells (SOX10‐EGFP). The results demonstrate significant neuronal Kir7.1 immunostaining in the cortex, hippocampus, cerebellum and pons, as well as the striatum and hypothalamus. In addition, astrocytes are shown to be immunopositive for Kir7.1 throughout grey and white matter, with dense immunostaining on cell somata, primary processes and perivascular end‐feet. Immunostaining for Kir7.1 was observed in oligodendrocytes, myelin and OPCs throughout the brain, although immunostaining was heterogeneous. Neuronal and glial expression of Kir7.1 is confirmed using neurone‐glial cortical cultures and optic nerve glial cultures. Notably, Kir7.1 have been shown to regulate the excitability of thalamic neurones and our results indicate this may be a widespread function of Kir7.1 in neurones throughout the brain. Moreover, based on the function of Kir7.1 in multiple transporting epithelia, Kir7.1 are likely to play an equivalent role in the primary glial function of K+ homeostasis. Our results indicate Kir7.1 are far more pervasive in the brain than previously recognised and have potential importance in regulating neuronal and glial function.
Highlights
There are seven subfamilies of Kir channels, Kir1–7, and each subfamily has multiple members that can form as homotetramers or heterotetramers (Lagrutta et al 1996; Pessia et al 1996; Rojas et al 2007)
Mouse strains used were: C57BL6/10 wild type; PLP-DsRed transgenic reporter mice, in which expression of DsRed is driven by the oligodendrocyte-specific proteolipid protein (PLP) gene promoter (Hirrlinger et al 2005); GFECEGFP transgenic reporter mice, in which expression of enhanced green fluorescent protein (EGFP) is driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) gene promoter (Nolte et al 2001); SOX10-EGFP transgenic reporter mice, in which expression of EGFP is driven by the oligodendrocyte lineage-specific SOX10 gene promoter (Matsuoka et al 2005; Kessaris et al 2006); and PdgfracreERT2-Rosa26-YFP mice, in which Cre recombination and YFP expression is driven by the oligodendrocyte progenitor cell (OPC)specific gene Pdgfra, induced by administration of tamoxifen, as previously detailed (Rivers et al 2008)
Western blot analysis of protein lysates from mouse cerebellum (CBL) and cortex (CTX) confirmed robust Kir7.1 protein expression with a predicted band at 54 kDa (Krapivinsky et al 1998; Ango et al 2004); positive bands were completely eliminated in the presence of the competitive peptide, which served as a further negative control (Fig. 1D)
Summary
There are seven subfamilies of Kir channels, Kir, and each subfamily has multiple members that can form as homotetramers or heterotetramers (Lagrutta et al 1996; Pessia et al 1996; Rojas et al 2007). Expression of Kir7.1 has been demonstrated in multiple transporting epithelia, in the small intestine (Partiseti et al 1998), choroid plexus (Doring et al 1998) gastric parietal cells (Fujita et al 2002; Malinowska et al 2004), kidney (Ookata et al 2000; Derst et al 2001; Suzuki et al 2003),. Kir7.1 transcript and protein expression has been demonstrated using in situ hybridisation, immunohistochemistry, Western and Northern blot, microarray and reverse transcription polymerase chain reaction (rtPCR) in human, rat, mouse, rabbit, guinea pig, bovine, porcine and monkey (Doring et al 1998; Ookata et al 2000; Derst et al 2001; Suzuki et al, 2003; Yasuda et al 2003; Pondugula et al 2006; Yang et al, 2003). We demonstrate widespread Kir7.1 immunostaining in neurones and glia throughout the adult mouse brain
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