Abstract
Inward rectifying potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis in oligodendrocytes, the myelinating cells of the central nervous system (CNS). Prominent expression of Kir4.1 has been indicated in oligodendrocytes, but the extent of expression of other Kir subtypes is unclear. Here, we used qRT-PCR to determine expression of Kir channel transcripts in the mouse optic nerve, a white matter tract comprising myelinated axons and the glia that support them. A novel finding was the high relative expression of Kir7.1, comparable to that of Kir4.1, the main glial Kir channel. Significantly, Kir7.1 immunofluorescence labelling in optic nerve sections and in isolated cells was localised to oligodendrocyte somata. Kir7.1 are known as a K+ transporting channels and, using patch clamp electrophysiology and the Kir7.1 blocker VU590, we demonstrated Kir7.1 channels carry a significant proportion of the whole cell potassium conductance in oligodendrocytes isolated from mouse optic nerves. Notably, oligodendrocytes are highly susceptible to ischemia/hypoxia and this is due at least in part to disruption of ion homeostasis. A key finding of this study is that blockade of Kir7.1 with VU590 compromised oligodendrocyte cell integrity and compounds oligodendroglial loss in ischemia/hypoxia in the oxygen–glucose deprivation (OGD) model in isolated intact optic nerves. These data reveal Kir7.1 channels are molecularly and functionally expressed in oligodendrocytes and play an important role in determining oligodendrocyte survival and myelin integrity.
Highlights
Oligodendrocytes myelinate axons in the central nervous system (CNS) and are essential for the rapid conduction of neural impulses
With subsequent multiple comparisons; qRT-Polymerase Chain Reactions (PCR) were ran in triplicate for each age group, with n = 10 optic nerves per run (n = 5 animals per qRT-PCR). b, c Confirmation of Kir7.1 expression in the optic nerve by comparison with brain, by room temperature (RT)-PCR (b) and Western blot (c), with detection of the expected Kir7.1 mRNA product at 251 bp (b) and protein at 54 kDa (c); no bands were detected in negative controls, in the absence of cDNA in the reaction mix for RT-PCR (b) and pre-incubation with the blocking peptide for western blots (c)
Due to very similar MW of the proteins, the same samples were run in parallel and under identical conditions on different western blots, using β-actin as a positive control to demonstrate the presence of Kir7.1 protein (Fig. 1b) and 54 kDa for protein (Fig. 1c) (Krapivinsky et al 1998; Pattnaik et al 2013); positive bands were absent in negative controls, in the absence of cDNA in the reaction mix for RT-PCR (Fig. 1b) and in the presence of the competitive peptide for western blot (Fig. 1d)
Summary
Oligodendrocytes myelinate axons in the central nervous system (CNS) and are essential for the rapid conduction of neural impulses. Loss of oligodendrocytes and subsequently myelin has devastating effects on CNS function, such as occurs in the demyelinating disease Multiple Sclerosis (MS). Oligodendroglial membrane potential is largely determined by plasmalemmal inward rectifying potassium channels (Kir) (Neusch et al 2001). Functional Kir channels are formed either through homomeric alpha subunit tetramerisation, or by heteromeric assembly, thereby increasing functional diversity (Hibino et al 2010). A prominent role for Kir4.1 channels has been demonstrated in myelination and maintaining WM integrity under pathological conditions (Neusch et al 2001; Bolton and Butt 2006; Schirmer et al 2018).
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