Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes

Department of Biochemistry, Universidad Central del Caribe, Bayamón, Puerto Rico.
Glia (Impact Factor: 6.03). 02/2007; 55(3):274-81. DOI: 10.1002/glia.20455
Source: PubMed

ABSTRACT Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.

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Available from: Serguei N Skatchkov, Jul 18, 2015
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    • "n potential can increase [ K 1 ] e by as much as 1 mM ( Ransom et al . , 2000 ) . Kir4 . 1 mediated K 1 buffering is a mechanism for removing this [ K 1 ] e , which requires no energy . The importance of Kir4 . 1 in brain and SC astrocytes , and its role in K 1 homeostasis , has been demonstrated using Kir4 . 1 tar - geted siRNA in rats and mice ( Kucheryavykh et al . , 2007 ) , and in situ using conventional and conditional knockout ani - mals ( Djukic et al . , 2007 ; Neusch et al . , 2001 ) . Collectively , these studies examining astrocytes from the cortex , hippocam - pus , SC and retina show astrocytes lacking Kir4 . 1 are devoid of inwardly rectifying current , have significantly increased input resi"
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    ABSTRACT: Kir4.1, a glial-specific K+ channel, is critical for normal CNS development. Studies using both global and glial-specific knockout of Kir4.1 reveal abnormal CNS development with the loss of the channel. Specifically, Kir4.1 knockout animals are characterized by ataxia, severe hypomyelination, and early postnatal death. Additionally, Kir4.1 has emerged as a key player in several CNS diseases. Notably, decreased Kir4.1 protein expression occurs in several human CNS pathologies including CNS ischemic injury, spinal cord injury, epilepsy, ALS, and Alzheimer's disease. Despite the emerging significance of Kir4.1 in normal and pathological conditions, its mechanisms of regulation are unknown. Here, we report the first epigenetic regulation of a K+ channel in the CNS. Robust developmental upregulation of Kir4.1 expression in rats is coincident with reductions in DNA methylation of the Kir4.1 gene, KCNJ10. Chromatin immunoprecipitation reveals a dynamic interaction between KCNJ10 and DNA methyltransferase 1 during development. Finally, demethylation of the KCNJ10 promoter is necessary for transcription. These findings indicate DNA methylation is a key regulator of Kir4.1 transcription. Given the essential role of Kir4.1 in normal CNS development, understanding the regulation of this K+ channel is critical to understanding normal glial biology. GLIA 2014.
    Glia 03/2014; 62(3). DOI:10.1002/glia.22613 · 6.03 Impact Factor
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    • "These comprise Kir4.1 channels, homo-tetramers of Kir4.1 subunits, and Kir4.1/5.1 channels, hetero-tetramers of Kir4.1 and Kir5.1 subunits, which conduct large inward K + currents at potentials negative to K + equilibrium potential (Tanemoto et al., 2000; Ohno et al., 2007; Su et al., 2007; Furutani et al., 2009). In addition, spatial K + buffering is linked to glutamate uptake and/or aquaporin-4-mediated water transport by astrocytes (Nagelhus et al., 1999; Amiry-Moghaddam and Ottersen, 2003; Puwarawuttipanit et al., 2006; Djukic et al., 2007; Kucheryavykh et al., 2007). "
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    ABSTRACT: The inwardly rectifying potassium (Kir) channel Kir4.1 in brain astrocytes mediates spatial K(+) buffering and regulates neural activities. Recent studies have shown that loss-of-function mutations in the human gene KCNJ10 encoding Kir4.1 cause epileptic seizures, suggesting a close relationship between the Kir4.1 channel function and epileptogenesis. Here, we performed expressional analysis of Kir4.1 in a pilocarpine-induced rat model of temporal lobe epilepsy (TLE) to explore the role of Kir4.1 channels in modifying TLE epileptogenesis. Treatment of rats with pilocarpine (350 mg/kg, i.p.) induced acute status epilepticus, which subsequently caused spontaneous seizures 7-8 weeks after the pilocarpine treatment. Western blot analysis revealed that TLE rats (interictal condition) showed significantly higher levels of Kir4.1 than the control animals in the cerebral cortex, striatum, and hypothalamus. However, the expression of other Kir subunits, Kir5.1 and Kir2.1, remained unaltered. Immunohistochemical analysis illustrated that Kir4.1-immunoreactivity-positive astrocytes in the pilocarpine-induced TLE model were markedly increased in most of the brain regions examined, concomitant with an increase in the number of glial fibrillary acidic protein (GFAP)-positive astrocytes. In addition, Kir4.1 expression ratios relative to the number of astrocytes (Kir4.1-positive cells/GFAP-positive cells) were region-specifically elevated in the amygdala (i.e., medial and cortical amygdaloid nuclei) and sensory cortex. The present study demonstrated for the first time that the expression of astrocytic Kir4.1 channels was elevated in a pilocarpine-induced TLE model, especially in the amygdala, suggesting that astrocytic Kir4.1 channels play a role in modifying TLE epileptogenesis, possibly by acting as an inhibitory compensatory mechanism.
    Frontiers in Cellular Neuroscience 07/2013; 7:104. DOI:10.3389/fncel.2013.00104 · 4.18 Impact Factor
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    • "In addition, spatial K + buffering is linked to glutamate uptake and/or aquaporin-4-mediated water transport by astrocytes (Amiry-Moghaddam and Ottersen, 2003; Djukic et al., 2007; Kucheryavykh et al., 2007; Nagelhus et al., 1999; Puwarawuttipanit et al., 2006). Previous studies have shown that down-regulation of Kir4.1 channels increases extracellular glutamate concentration and neural excitability, suggesting a close relationship between Kir4.1 dysfunction and epileptogenesis (Djukic et al., 2007; Kucheryavykh et al., 2007). In addition, recent clinical studies demonstrated that mutations in the human gene KCNJ10 encoding Kir4.1 induce EAST (or SeSAME) syndrome including generalized tonic-clonic (GTC) seizures and ataxia (e.g., unstable gait and/or frequent falls), hearing loss and abnormal renal excretion of electrolytes (e.g., increased potassium excretion and hypokalemia) (Bockenhauer et al., 2009; Scholl et al., 2009). "
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    ABSTRACT: The inwardly rectifying potassium channel subunit Kir4.1 is expressed in brain astrocytes and involved in spatial K(+) buffering, regulating neural activity. To explore the pathophysiological alterations of Kir4.1 channels in epileptic disorders, we analyzed interictal expressional levels of Kir4.1 in the Noda epileptic rat (NER), a hereditary animal model for generalized tonic-clonic (GTC) seizures. Western blot analysis showed that Kir4.1 expression in NERs was significantly reduced in the occipito-temporal cortical region and thalamus. However, the expression of Kir5.1, another Kir subunit mediating spatial K(+) buffering, remained unaltered in any brain regions examined. Immunohistochemical analysis revealed that Kir4.1 was primarily expressed in glial fibrillary acidic protein (GFAP)-positive astrocytes (somata) and foot processes clustered around neurons proved with anti-neuronal nuclear antigen (NeuN) antibody. In NERs, Kir4.1 expression in astrocytic processes was region-selectively diminished in the amygdaloid nuclei (i.e., medial amygdaloid nucleus and basomedial amygdaloid nucleus) while Kir4.1 expression in astrocytic somata was unchanged. Furthermore, the amygdala regions with reduced Kir4.1 expression showed a marked elevation of Fos protein expression following GTC seizures. The present results suggest that reduced activity of astrocytic Kir4.1 channels in the amygdala is involved in limbic hyperexcitability in NERs.
    Brain research 04/2013; 1517. DOI:10.1016/j.brainres.2013.04.009 · 2.83 Impact Factor
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