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


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, Oct 04, 2015
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    • "Freshly dissociated hippocampal tissues as a new model for study of astrocyte function. The use of acutely isolated astrocytes has generally been considered a highly valuable model to gain insight into the basic property and function of astrocytes (Kimelberg et al. 2000). During development, astrocyte gene expression and function change dramatically (Cahoy et al. 2008; Sun et al. 2013). "
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    ABSTRACT: Mature astrocytes exhibit a linear current-to-voltage (I-V) relationship K(+) membrane conductance (passive conductance) and an extremely low membrane resistance (RM) in situ. The combination of these electrophysiological characteristics establishes a highly negative and stable membrane potential that is essential for the basic functions, such as K(+) spatial buffering and neurotransmitter uptake. However, astrocytes are coupled extensively in situ. It remains to be determined whether the observed passive behavior and low RM are attributable to the intrinsic properties of membrane ion channels or to gap junction coupling in functionally mature astrocytes. In the present study, freshly dissociated hippocampal tissues were used as a new model to examine this basic question in young adult animals. The morphologically intact single astrocytes could be reliably dissociated from P21 and older animals. At this animal age, dissociated single astrocytes exhibit a similar passive conductance and resting membrane potential as astrocytes do in situ. To precisely measure the RM from single astrocytes, dual patch single astrocyte recording was performed. We show that dissociated single astrocytes exhibit a similarly low RM as syncytial coupled astrocytes. Functionally, the symmetric expression of high K(+) conductance enabled rapid change in the intracellular K(+) concentrations in response to changing K(+) driving force. Altogether, we demonstrate that freshly dissociated tissue preparation is a highly useful model for study of the functional expression and regulation of ion channels, receptors, and transporters in astrocytes, and the passive behavior and low RM are the intrinsic properties of mature astrocytes. Copyright © 2015, Journal of Neurophysiology.
    Journal of Neurophysiology 03/2015; 113(10):jn.00206.2015. DOI:10.1152/jn.00206.2015 · 2.89 Impact Factor
    • "Among these membrane proteins, Kir channels play a critical role, especially homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels. The Kir4.1 channel is an important player in maintaining the hyperpolarized membrane potential of astrocytes (Kucheryavykh et al., 2007). The dysfunctions of these channels result in neurological disorders including epilepsy. "
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    ABSTRACT: Epilepsy is a neurological disorder that affects around 1% of the population worldwide. The two main therapies, pharmacology and the electrical stimulation, both have some shortcomings. For instance, pharmacological therapy is frequently accompanied by side effects, and current anticonvulsive drugs fail to be effective to around a third of patients. These patients could suffer astrocyte-related epilepsy, as increasing evidence indicates that dysfunctions of astrocytes can result in epilepsy. However, epilepsy drugs that affect astrocytes are not available currently. Although electrical stimulation has benefited many patients, the electrode stimulates unselective neurons or circuits. All these need to develop new strategies for improving the life of the patients. As channelrhodopsins (ChRs) were discovered, a novel method referred to as “optogenetics” was developed. It has advantages over electrical stimulation of being less-invasiveness and allowing spatiotemporally stimulation. Recently, a number of experiments have explored the treatments for epilepsy with optogenetic control of neurons. Here, we discuss the possibility that an optogenetic approach could be used to control the release of gliotransmitters and improve astrocyte function such as glutamate and K+ uptake, and thereby offer a potential strategy to investigate and treat astrocyte-related epilepsy.
    Brain Research Bulletin 11/2014; 110. DOI:10.1016/j.brainresbull.2014.10.013 · 2.72 Impact Factor
    • "The inwardly rectifying potassium channels Kir4.1 abundantly expressed in astrocytes contribute to K + spatial buffering, a fundamental mechanism in maintaining neuronal excitability and synaptic transmission (Olsen and Sontheimer, 2008), and are implicated in the regulation of cell volume (Benesova et al., 2012; Haj-Yasein et al., 2011; Obara-Michlewska et al., 2011; Pannicke et al., 2006). Downregulation of Kir4.1 channels expression has been reported to decrease glutamate (Glu) uptake in cultured astrocytes (Kucheryavykh et al., 2007) and in astrocytes in hippocampal slices (Djukic et al., 2007). Kir4.1 channels were found to control swelling of astroglial processes during ischaemia in the experimental spinal cord oedema (Dibaj et al., 2007). "
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    ABSTRACT: Astroglial inward rectifying Kir4.1 potassium channels are fundamental for the maintenance of ion and water homeostasis in the central nervous system (CNS). Down-regulation of Kir4.1 expression is observed in CNS disorders associated with excessive extracellular glutamate (Glu) accumulation, including hepatic encephalopathy related to acute liver failure (ALF). Here we demonstrate that prolonged (3 days) treatment of cultured rat cortical astrocytes with 2 mM Glu or 100 µM NMDA decreases the expression of Kir4.1 mRNA and protein. Inhibition by Glu of Kir4.1 mRNA expression was reversed by NMDA receptor antagonists MK-801 and AP-5 (each at 50 µM), and by a non-transportable inhibitor of Glu uptake TBOA (100 µM). MK-801 reversed the inhibitory effect of Glu on Kir4.1 protein expression. In contrast, transcription of Kir4.1 channels was not affected by: (i) a transportable Glu uptake inhibitor PDC (100 µM); (ii) by group I mGluR antagonist MTEP (100 µM); (iii) by antagonists of oxidative-nitrosative stress (ONS) in astrocytes, including the neuroprotective amino acid taurine (Tau; 10 mM), the NADPH oxidase inhibitor apocyanine (APO; 300 µM), the nitric oxide synthase inhibitor, L-NNA (100 µM), and a membrane permeable glutathione precursor, glutathione-diethyl ester (GEE; 3 mM). Down-regulation of Kir4.1 transcription in rats with ALF was attenuated by intraperitoneal administration of a competitive NMDA receptor antagonist memantine, but not by histidine, which reverses ONS associated with ALF. Collectively, the results indicate that over-activation of astroglial NMDA receptors, aided by as yet undefined effects of Glu entry to astrocytes, is a primary cause of the reduction of Kir4.1 expression in CNS disorders associated with increased exposure to Glu.
    Neurochemistry International 10/2014; 88. DOI:10.1016/j.neuint.2014.10.006 · 3.09 Impact Factor
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