Functional implications for Kir4.1 channels in glial biology: from K+ buffering to cell differentiation

Abstract

Astrocytes and oligodendrocytes are characterized by a very negative resting potential and a high resting permeability for K(+) ions. Early pharmacological and biophysical studies suggested that the resting potential is established by the activity of inwardly rectifying, Ba(2+) sensitive, weakly rectifying Kir channels. Molecular cloning has identified 16 Kir channels genes of which several mRNA transcripts and protein products have been identified in glial cells. However, genetic deletion and siRNA knock-down studies suggest that the resting conductance of astrocytes and oligodendrocytes is largely due to Kir4.1. Loss of Kir4.1 causes membrane depolarization, and a break-down of K(+) and glutamate homeostasis which results in seizures and wide-spread white matter pathology. Kir channels have also been shown to act as critical regulators of cell division whereby Kir function is correlated with an exit from the cell cycle. Conversely, loss of functional Kir channels is associated with re-entry of cells into the cell cycle and gliosis. A loss of functional Kir channels has been shown in a number of neurological diseases including temporal lobe epilepsy, amyotrophic lateral sclerosis, retinal degeneration and malignant gliomas. In the latter, expression of Kir4.1 is sufficient to arrest the aberrant growth of these glial derived tumor cells. Kir4.1 therefore represents a potential therapeutic target in a wide variety of neurological conditions.

Fig. 1

Signature features of astrocytic inward rectifiers: (a) Representative recordings from spinal cord astrocytes demonstrating currents that activate with negative voltage steps. Current amplitude increases markedly with elevated extracellular K⁺ (control 5 mM). At the most negative voltages currents show time-dependent inactivation because of a block by extracellular Mg²⁺. Inward currents are completely inhibited by 100 μM Ba²⁺. (b) A continuous ramp-like change in voltage ranging from −150 mM to 150 mV was used before and after application of Ba²⁺, and the subtracted Ba²⁺ sensitive current was plotted in (b) and (c). The resulting current-voltage curve was weakly rectifying in (b) recorded from a spinal cord astrocytes after 8 days in culture, showing smaller outward currents than inward currents. The same approach was taken in (c) in a recording from a microglial cells. The resulting current-voltage curve showed strong rectification with essentially no outward current and only inward currents at potentials negative of the E_k. [With permission from Olsen et al. 2006].

Fig. 2

Inwardly rectifying K⁺ currents in astrocytes are mediated by Kir4.1: (a) Voltage-ramps were used to elicit currents in astrocytes isolated from wildtype (WT,1) and Kir4.1 knockout (KO,2) animals. Only wildtype recordings showed any significant inward currents. (b) Using voltage step protocols, wildtype astrocytes showed pronounced inward currents with characteristic inactivation at negative potentials. Inward currents were completely absent in astrocytes from Kir4.1 knockout animals. (c) Astrocytes cultured from knock-out animals demonstrated a near 5-fold increase in input resistance. (d) Resting membrane potential is significantly depolarized in astrocytes cultured from knock out animals (*p < 0.05). [With permission from Olsen et al. 2006].

Fig. 3

Loss of Kir4.1 inhibits K⁺ and glutamate uptake. (a,b) Patch-clamp recordings were obtained in astrocytes in slices from the ventral respiratory group in wildtype and Kir4.1 knockout animals. Application of 50 mM K⁺ induced an inward current in wildtype astrocytes but not in astrocytes from Kir4.1 knockout mice (*p < 0.05). [(a,b) With permission from Neusch et al. 2006; (c,d) With permission from Djukic et al. 2007].

Fig. 4

Kir4.1 is developmentally regulated in glia in the spinal cord. (a) Images from the ventral horn demonstrating Kir 4.1 largely overlapped with GLT-1 as demonstrated by the yellow color in the merged image. (b) Kir4.1 and Neu-N labeling appear distinct in the ventral horn. (c) High magnification images from the ventral horn demonstrate Kir4.1 staining is most intense surrounding neuronal cell bodies. [Scale bars (a) and (b), 100 μm, (c) 50μm]. (d) Whole-cell lysates from wild-type rat spinal cord were probed with antibodies to Kir4.1 and two bands at 55 kD and ~200 kD characteristic of monomeric and tetrameric Kir4.1 alpha subunits. Expression was very weak at P0, increased notably at P15 and was high at P30 suggesting a developmental gain in expression of Kir4.1 in spinal cord. (e) Over the same developmental time period, recordings with extracellular K⁺ electrodes in optic nerve in situ suggest a tightening of extracellular K⁺ fluctuations with developmental age from P2 to adulthood. [(a–c) With permission from Olsen et al., 2007; (d) With permission from Olsen et al. 2006; (e) with permission from Connors et al. 1982].

Fig. 5

Role of Kir4.1 in glial growth control: (a) Immunostaining with antibodies to Kir4.1 and the cytoskeleton with Phalloiden (Kir4.1: green, Phalloiden:red) show Kir4.1 labels the cell nucleous. (b) In contrast in astrocytes Kir4.1 (green) labels the cell membrane (the cytoskeleton is labeled with GFAP, red). (c) This difference is also reflected in patch-clamp recordings from another glioma cells line (D54) which lack inward currents compared to SC astrocytes that express prominent inward currents. However, upon transfection of D54MG cells with a GFP containing plasmid encoding Kir4.1, these glioma cells express inward currents that are indistinguishable from astrocytic Kir currents. (d) Expression of Kir4.1 in glioma caused their resting potential to shift ~30 mV negative making it similar to that of differentiated astrocytes. (e) The presence or absence of functional Kir4.1 channels determined the rate of growth of D54 glioma cells, in which growth was retarded when Kir4.1 channels were functional. However, depolarizing the cell with 20 mM K⁺ was sufficient to rescue growth even when Kir4.1 was over-expressed [(a) With permission from Olsen and Sontheimer 2004a; (b) With permission Olsen et al. 2006; (c–e) With permission from Higashimori and Sontheimer 2007].