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
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    • "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.
    Full-text · Article · Oct 2015 · Neurochemistry International
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    • "Glutamate clearance by astrocytes grown in high glucose is impaired Because decreased expression of Kir4.1 channel subunits has been associated with decreased glutamate clearance by astrocytes (Djukic et al., 2007; Kucheryavykh et al., 2007), we next examined the glutamate uptake capability of astrocytes cultured in control or high glucose using a colorimetric assay (Abe et al., 2000). We found that astrocytes grown in DMEM containing high glucose (25 mM) clear less glutamate than astrocytes grown in DMEM with 5 mM glucose. "
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    ABSTRACT: Diabetics are at risk for a number of serious health complications including an increased incidence of epilepsy and poorer recovery after ischemic stroke. Astrocytes play a critical role in protecting neurons by maintaining extracellular homeostasis and preventing neurotoxicity through glutamate uptake and potassium buffering. These functions are aided by the presence of potassium channels, such as Kir4.1 inwardly rectifying potassium channels, in the membranes of astrocytic glial cells. The purpose of the present study was to determine if hyperglycemia alters Kir4.1 potassium channel expression and homeostatic functions of astrocytes. We used q-PCR, Western blot, patch-clamp electrophysiology studying voltage and potassium step responses and a colorimetric glutamate clearance assay to assess Kir4.1 channel levels and homeostatic functions of astrocytes grown in normal and high glucose conditions. We found that astrocytes grown in high glucose (25mM) had an approximately 50% reduction in Kir4.1 mRNA and protein expression as compared with those grown in normal glucose (5mM). These reductions occurred within 4-7days of exposure to hyperglycemia, whereas reversal occurred between 7 and 14days after return to normal glucose. The decrease in functional Kir channels in the astrocytic membrane was confirmed using barium to block Kir channels. In the presence of 100-μm barium, the currents recorded from astrocytes in response to voltage steps were reduced by 45%. Furthermore, inward currents induced by stepping extracellular [K(+)]o from 3 to 10mM (reflecting potassium uptake) were 50% reduced in astrocytes grown in high glucose. In addition, glutamate clearance by astrocytes grown in high glucose was significantly impaired. Taken together, our results suggest that down-regulation of astrocytic Kir4.1 channels by elevated glucose may contribute to the underlying pathophysiology of diabetes-induced CNS disorders and contribute to the poor prognosis after stroke.
    Full-text · Article · Sep 2015 · Neuroscience
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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.
    Full-text · Article · Mar 2015 · Journal of Neurophysiology
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