Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes.
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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ABSTRACT: Epilepsy is a prevalent neurological disorder afflicting nearly 50 million people worldwide. The disorder is characterized clinically by recurrent spontaneous seizures attributed to abnormal synchrony of brain neurons. Despite advances in the treatment of epilepsy, nearly one-third of patients are resistant to current therapies, and the underlying mechanisms whereby a healthy brain becomes epileptic remain unresolved. Therefore, researchers have a major impetus to identify and exploit new drug targets. Here we distinguish between epileptic effectors, or proteins that set the seizure threshold, and epileptogenic mediators, which control the expression or functional state of the effector proteins. Under this framework, we then discuss attempts to regulate the mediators to control epilepsy. Further insights into the complex processes that render the brain susceptible to seizures and the identification of novel mediators of these processes will lead the way to the development of drugs to modify disease outcome and, potentially, to prevent epileptogenesis. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 55 is January 06, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.Annual Review of Pharmacology 08/2014; · 18.52 Impact Factor
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ABSTRACT: The inwardly rectifying potassium (Kir) channel subunit Kir4.1 is specifically expressed in brain astrocytes and Kir4.1-containing channels (Kir4.1 channels) mediate astroglial spatial potassium (K +) buffering. Recent advances in Kir4.1 research revealed that Kir4.1 channels can serve as a novel therapeutic target for epilepsy. Specifically, reduced expression or dysfunction of Kir4.1 channels seems to be involved in generation of generalized tonic-clonic seizures (GTCS) in animal models of epilepsy and patients with temporal lobe epilepsy. In addition, recent clinical studies showed that loss-of-function mutations of human gene (KCNJ10) encoding Kir4.1 elicit " EAST " or " SeSAME " syndrome which manifests as GTCS and ataxia. Although the precise mechanisms remain to be clarified, it is suggested that dysfunction of Kir4.1 channels disrupts spatial K + buffering by astrocytes, elevates extracellular levels of K + and/or glutamate and causes abnormal excitation of neurons in the limbic regions and neocortex. All these findings suggest that agents that activate or up-regulateastroglialKir4.1 channels would be effective for epilepsy. In addition, docking simulation analysis usingtheKir4.1 homology model provide simportant information for designing new Kir4.1 ligands. Discovery of suchagents that activate or up-regulate Kir4.1 channels would be a novel approach for the treatment of epilepsy.Ther Targets Neurol Dis. 01/2015; 2(e476):1-10.
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ABSTRACT: Glutamate (Glu) and NMDA decrease Kir4.1 expression in cultured astrocytes.•Glu-induced loss of Kir4.1 is reversed by NMDA receptor antagonists (NMDAra).•Loss of brain Kir4.1 mRNA in hepatic encephalopathy is attenuated by a NMDAra.•Results implicate astrocytic NMDA receptors in Kir4.1 loss in Glu-overexposed brain.Neurochemistry International 10/2014; · 2.65 Impact Factor
Downregulation of Kir4.1 Inward Rectifying Potassium
Channel Subunits by RNAi Impairs Potassium Transfer
and Glutamate Uptake by Cultured Cortical Astrocytes
Y. V. KUCHERYAVYKH,1L. Y. KUCHERYAVYKH,1C. G. NICHOLS,2H. M. MALDONADO,3K. BAKSI,4
A. REICHENBACH,5S. N. SKATCHKOV,1,6AND M. J. EATON1*
1Department of Biochemistry, Universidad Central del Caribe, Bayam? on, Puerto Rico
2Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri
3Department of Pharmacology, Universidad Central del Caribe, Bayam? on, Puerto Rico
4Department of Anatomy, Universidad Central del Caribe, Bayam? on, Puerto Rico
5Paul Flechsig Institute of Brain Research, Leipzig University, Leipzig, Germany
6Department of Physiology, Universidad Central del Caribe, Bayam? on, Puerto Rico
K1buffering; glutamate clearance; Kcnj10; RNA interference
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 abnor-
mal synaptic transmission. The role of the different K1chan-
nels in maintaining the membrane potential and buffering
capabilities of cortical astrocytes has not yet been definitively
determined due to the lack of specific K1channel blockers.
The purpose of the present study was to assess the role of the
inward-rectifying K1channel 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
268 and 241 mV. This distribution became unimodal after
knockdown of Kir4.1, with the mean membrane potential
being shifted in the depolarizing direction (peak at 245 mV).
The ability of Kir4.1-suppressed cells to mediate transmem-
brane potassium flow, as measured by the current response
to voltage ramps or sequential application of different extra-
cellular [K1], was dramatically impaired. In addition, gluta-
mate uptake was inhibited by knock-down of Kir4.1-contain-
ing channels by RNA interference as well as by blockade of
Kir channels with barium (100 lM). 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 hyperpolar-
ization in this process.
C2006 Wiley-Liss, Inc.
Astrocytes regulate the extracellular concentrations of
neuroactive substances such as K1, H1, GABA, and gluta-
mate and thereby, modulate both neuronal excitability
and synaptic transmission. The membrane potential of
astrocytic glial cells is generally considerably more hyper-
polarized than that of neurons, and is maintained by a
wide variety of potassium channels (Verkhratsky and
Steinh€ auser, 2000). One of the major classes of potassium
channels in glial cells is the inward-rectifiers (i.e. the Kir
subfamily) (Biedermann et al., 1998; Brew et al., 1986;
Higashi et al., 2001; Kofuji et al., 2000; Li et al., 2001;
Skatchkov et al., 1995; Sontheimer, 1994). In the specia-
lized M€ uller glial cells of the retina, the channel proteins
underlying their K1conductances have been intensely
studied (Bringmann et al., 1997; Ishii et al., 1997; Raap
et al., 2002; Skatchkov et al., 2001, 2002, 2006a). In the
retina, Kir4.1 channels are mainly found in M€ uller glial
cells (Dalloz et al., 2003; Ishii et al., 1997; Kofuji et al.,
2002; Nagelhus et al., 1999; Skatchkov et al., 2001). In
mice in which Kir4.1 subunits were genetically inacti-
vated, the resting membrane potentials of the M€ uller cells
were significantly depolarized, indicating a dominant role
for Kir4.1 channels in setting the membrane potential
(Kofuji et al., 2000). In these mice, spatial buffering K1-
currents through M€ uller cells into the subretinal space
were dramatically reduced (Kofuji et al., 2000). A similar
decrease of K1buffering currents was suggested to occur
in M€ uller cells of postischemic rats in which the expres-
sion of Kir4.1 channels is impaired (Pannicke et al., 2004).
In spinal cord astrocytes, Kir4.1 channels also appear to
be responsible for the major resting K1conductance
(Olsen et al., 2006). In the brain, however, Kir4.1 subunits
display a heterogeneous distribution (Higashi et al., 2001;
Li et al., 2001; Poopalasundaram et al., 2000; Schr€ oder
et al., 2002; Takumi et al., 1995) and Kir4.1 protein, as
well as Kir4.1 transcripts, were found in only about 50%
The contents of this article are solely the responsibility of the authors and do not
necessarily represent the official views of National Center for Research Resources or
National Institute of Health.
Grant sponsor: NIH; Grant numbers: NIH-NINDS-S11-NS48201, NIH-MBRS-
SO6-GM50695, NIH-NCRR-RCMI-G12RR03035; Grant sponsor: Interdisziplin€ ares
Zentrum f€ ur Klinische Forschung, Medical Faculty of the Universit€ at Leipzig (pro-
*Correspondence to: Misty J. Eaton, Department of Biochemistry, Universidad
Central del Caribe, P.O. Box 60-327, Bayam? on, Puerto Rico 00960-6032.
Received 29 March 2006; Revised 20 September 2006; Accepted 4 October 2006
Published online 7 November 2006 in Wiley InterScience (www.interscience.
GLIA 55:274–281 (2007)
C2006 Wiley-Liss, Inc.
of brain astrocytes (Higashi et al., 2001; Schr€ oder et al.,
2002). Although the distribution of Kir4.1 subunits near
synapses and blood vessels suggests a role in K1buffering
(Higashi et al., 2001), a number of other types of K1chan-
nel subunits were found in brain astrocytes (for review
see Verkhratsky and Steinh€ auser, 2000). Therefore, it re-
mains to be clarified which type(s) of K1channels provide
the major membrane potential and the K1buffering cap-
abilities in cortical astrocytes.
A second major function of astrocytes is to clear gluta-
mate from the synaptic clefts. Glutamate clearance de-
pends upon a hyperpolarized membrane potential of the
glial cells involved (Brew and Attwell, 1987) and upon
intra- and extracellular potassium concentrations. When
glial cells are depolarized and the extracellular concen-
tration of potassium is elevated, the driving force for the
glutamate transporter is reduced, and extracellular glu-
tamate accumulates. Therefore, K1channels are not only
responsible for potassium homeostasis, but also are key
players in glutamate clearance.
In order to probe the role of different K1channel subu-
nits in control of membrane potential and spatial buffer-
ing by cortical astrocytes, the present study made use of
the novel approach of siRNA knock-down to specifically
suppress Kir4.1 channel subunit expression. The results
demonstrate effective use of this strategy and dramatic
consequences of Kir4.1 suppression, highlighting a critical
role of this subunit in K1conductance of cortical astro-
cytes. Some of the results of this study have been pre-
sented in abstract form (Eaton et al., 2005).
MATERIALS AND METHODS
Astrocyte Primary Cultures
Primary cultures of astrocytes were prepared from neo-
cortex of 1–2-day-old rats. Brains were removed after
decapitation and the meninges stripped away to mini-
mize fibroblast contamination. The forebrain cortices
were collected and dissociated using the stomacher
blender method. The cell suspension was then allowed to
filter by gravity through a no. 60 sieve and then through
a no. 100 sieve. After centrifugation, the cells were sus-
pended in modified Eagle’s medium (containing 30 mM
glucose, 2 mM glutamine, 1 mM pyruvate and 10% fetal
bovine serum, 100 units/mL of penicillin/streptomycin)
and plated in uncoated 75 cm2flasks at a density of
300,000 cells/cm2. The medium was exchanged with fresh
culture medium about every 5 days. At confluence (about
12–14 days), the mixed glial cultures were treated with
50 mM leucine methylester (pH 7.4) for 60 min to effec-
tively kill microglia (Simmons and Murphy, 1992). Cul-
tures were then allowed to recover for at least 1 day in
growth medium prior to experimentation. Astrocytes
were dissociated by trypsinization and reseeded onto the
appropriate plates or coverglasses for the experiments.
The purity of the astrocyte cultures was >95% as assessed
by immunocytochemical staining for glial fibrillary acidic
protein (GFAP; data not shown).
Transfection of siRNA Duplexes
RNA duplexes of 21 nucleotides specific for rat Kir4.1
(Kcnj10) were designed and chemically synthesized by
Qiagen (Valencia, CA). The Kir4.1 sense strand was GAG-
AUAGCACCGUACGUUAdTdT. The siRNA construct was
designed by Qiagen using their propietary HiPerformance
algorithm. The homology analysis tool used during design
of HP Guaranteed siRNA detects short regions of homol-
ogy, making it highly suitable for siRNA design and far
superior to BLAST homology searches, which often ex-
clude short regions of homology that could affect siRNA
Astrocytes were seeded at a density of 50,000–60,000
cells per well of a 24-well plate on the day of the transfec-
tion in 0.5 mL of growth medium containing 10% serum
and antibiotics. The cells were transfected using HiPer-
fect transfection reagent (Qiagen) and the Fast-Forward
Protocol for Transfection of Adherent Cells with siRNA
recommended by the manufacturer. Briefly, siRNA was
diluted in 100 lL culture medium without serum to give
a final concentration of 5 or 10 nM. Three microliters of
HiPerfect was added and the samples were incubated for
5–10 min at room temperature to allow the formation of
transfection complexes. The complexes were then added
to the cells in a drop-wise fashion and the plate gently
swirled to evenly distribute the transfection complexes.
Cells were incubated under normal growth conditions
until used (3–5 days later).
SDS-PAGE and Western Blotting Analysis
Astrocytes were harvested, pelleted, and stored at 280?C
until assayed. Pellets were sonicated briefly in ice-cold ho-
mogenization buffer (pH 7.5) containing: (in mM) Tris-
HCl 20, NaCl 150, MgCl210, EDTA 1.0, EGTA 1.0, PMSF
1.0, 1% Triton X-100, and an additional mixture of peptide
inhibitors (leupeptin, antipain, bestatin, chymostatin,
pepstatin each at a final concentration of 1.6 lg/mL). Pro-
tein concentration of cell homogenates was determined
with DC protein assay (Bio-Rad), followed by addition of
an appropriate volume of Urea sample buffer (62 mM
Tris/HCl pH 6.8, 4% SDS, 8 M Urea, 20 mM EDTA, 5% b-
Mercaptoethanol, 0.015% Bromophenol Blue) for a final
concentration of 0.5–1.5 lg protein/lL, and incubation in
a water bath at 100?C for 10 min. Samples were loaded
(10 lg/lane) onto 10% SDS-polyacrylamide gels (Protean
mini-gel system, Bio-Rad Laboratories, Hercules, CA),
and run for 45 min (200V; constant). After electrophoresis,
separated proteins were transferred overnight (12.5 h,
4?C) to a PVDF membrane with a Mini Trans-Blot appara-
tus (12V; constant) (Bio-Rad Laboratories, Hercules, CA)
and immediately stained with India ink. Subsequently,
the membranes were incubated overnight with blocking
solution containing: 5% BSA in 10 mM Tris, 100 mM
NaCl, 0.1% Tween 20 (TBST, pH 7.5). Blocked membranes
were then incubated for 2 h at room temperature with the
corresponding primary antibody (anti-Kir4.1, 1:400 dilu-
tion; Alomone Laboratories, Jerusalem), followed by three
Kir4.1 AND ASTROCYTIC K+AND GLUTAMATE BUFFERING
GLIA DOI 10.1002/glia
washes with TBST for 15 min and incubation with anti-
rabbit secondary antibody for 1 h. Final detection was per-
formed with enhanced chemiluminescence methodology
(SuperSignal? West Dura Extended Duration Substrate;
Pierce, Rockford, IL) as described by the manufacturer,
and the intensity of the signal measured in a gel documen-
tation system (Versa Doc Model 1000, Bio Rad). In all
cases, intensity of the chemiluminescence signal was cor-
rected for minor changes in protein content after densi-
tometry analysis of the India ink stained membrane.
Real Time RT-PCR
Validation of down-regulation of target gene expression
was performed by RT-PCR analysis for the Kcnj10 (Kir4.1)
RNA transcript. Two hundred thousand cells (4 wells with
50,000 cells each) were transfected as described above in
the presence and absence of 10 nM siRNA (Qiagen), and
incubated for 1–3 days. Total RNA (in 50 lL of water) was
isolated using an RNeasy Kit (Qiagen). Ten microliters of
each RNA sample was used for the determination of
mRNA using a LightCycler instrument (Roche Diagnos-
tics Indianapolis, IN) and a LightCycler Amplification kit
SYBR green 1 (Roche Diagnostics GmbH, Mannheim Ger-
many). Typical amplification reactions (20 lL) contained
3 mM MgCl2, 0.5 lM of each primer (validated sense and
antisense primers for the Kcnj10 gene; Qiagen) and 2 lL
of 10X LightCycler FastStart DNA Master SYBR Green I
(Roche Diagnostics). PCR amplifications were carried out
in glass capillary tubes (Roche Diagnostics). Reverse tran-
scription was performed at 55?C for 600 s, and amplifica-
tion began with a 30 s denaturation step at 95?C followed
by 45 cycles of annealing at 55?C for 10 s and extension at
72?C for 13 s. The standard curve for the mRNA was
determined using the LightCycler Control Kit (Roche
Diagnostics). The data were analyzed using LightCycler
Software (Roche Molecular Biochemicals).
Membrane currents and membrane potential were mea-
sured with the single electrode whole-cell patch-clamp
technique. Two Narishige hydraulic micromanipulators
(Narishige, MMW-203, Japan) were used for (1) voltage-
clamp recording, and (2) positioning a micropipette with
30–50 lm tip diameter for application of test solutions. A
5-valve system for fast drug application (MS Concentra-
tion Clamp; VS-2001, Vibraspec, PA), controlled by a sec-
ond computer, was connected to the outlets.
Electrodes from hard glass (GC-150-10 glass tubing,
Clark Electromedical Instruments, England) were pulled
in four steps using a Sutter P-97 puller (Novato, CA). After
filling with intracellular solution (ICS) containing (in mM):
KCl 141, MgCl21, CaCl21, EGTA 10, HEPES 10, Na2ATP
3, spermine HCl 0.25, pH adjusted to 7.2 with NaOH/HCl,
they had resistances of 4–6 MX; after cell penetration, the
access resistance was 10–15 MX, compensated by at least
75%. The extracellular solution (ECS) contained (in mM):
NaCl 138, CaCl22, MgCl21.9, and HEPES 10; KCl varied
from 1 to 30 mM (substituted by NaCl to adjust osmolarity
to 308 mOsm). High frequencies (>1 kHz) were cut off,
using an Axopatch-200B amplifier and a CV-203BU head-
stage, and digitized at 5 kHz through a DigiData 1200A
interface (Axon Instruments, USA). The pClamp 7 and 9
(Axon Instruments) software packages were used for data
acquisition and analysis.
The membrane potentials were determined immediately
after attainment of whole-cell mode, and cells were then
subsequently held under voltage-clamp at this potential.
Application of solutions with increased K1were used to
assess potassium uptake ability of the cell as the shift in
inward current following shift in [K1]ofrom 3 mM to 10 or
30 mM (Skatchkov et al., 1999).
Colorimetric Assay to Assess Glutamate
Clearance by Astrocytes
To evaluate the glutamate clearance capacity, astrocyte
cultures were grown in 24-well dishes and transfected as
described above. Glutamate remaining in the medium was
determined using the protocol of Abe and coworkers (Abe
and Misawa, 2003; Abe et al., 2000). Briefly, media in each
well of the dish was replaced with 300 lL of serum free
media containing 400 lM glutamate. After 60 min, the
media was removed and 50 lL of culture supernatant was
transferred to 96-well culture plates, and mixed with 50 lL
of substrate mixture (20 U/mL glutamate dehydrogenase,
2.5 mg/mL b-nicotineamide adenine dinucleotide (NAD),
zolium (MTT), 100 lM 1-methoxyphenazine methosulfate
(MPMS), 0.1% (v/v) Triton X-100 and 0.2 M Tris-HCl (pH
8.2)). Glutamate dehydrogenation was allowed to proceed
for 10 min at 37?C and then stopped by adding 100 lL of a
solution containing 50% dimethylformamide and 20% so-
azan was assessed by measurement of absorbance at
550 nm using a microplate reader. A standard curve was
tion of extracellular glutamate in the samples was esti-
mated from the standard curve. As a control for each
experiment, serum-free media containing 400 lM gluta-
mate was added to empty wells of a 24-well dish (no astro-
cytes) and processed together with the astrocytes, that is,
60 min in the incubator until sample collection. In all ex-
periments described in Fig. 4, the concentration of gluta-
mate in dishes with no astrocytes remained at ?400 lM.
After transfection with Kir4.1 siRNA, the levels of
Kir4.1 mRNA were analyzed by real time PCR. Mock-
transfected (transfected with HiPerfect and no siRNA)
astrocytes plated at a density of 50,000 cells/well con-
tained 9.09 3 1023pg of Kir4.1 mRNA in the assayed
sample at 3 days. Kir4.1 mRNA levels were significantly
KUCHERYAVYKH ET AL.
GLIA DOI 10.1002/glia
decreased by 92–95% on 1, 2, and 3 days after transfection
with siRNA as compared with mock-transfected astrocytes
plated at the same density (0.59 3 1023, 0.72 3 1023, and
0.41 3 1023pg at 1, 2, and 3 days, respectively). This cor-
responded with a strongly decreased Kir4.1 protein con-
tent as measured by Western blot (Fig. 1). The band at
50 kDa represents the monomer. In addition, we routinely
observed a band at 210 kDa that represents the tetramer
and was similarly reduced after siRNA treatment. The
electrophoretic mobility of Kir4.1 protein was similar to
that observed by Olsen et al. (2006).
Cultured cortical astrocytes (McKhann et al., 1997)
and freshly isolated hippocampal astrocytes (Zhou and
Kimelberg, 2000) have been reported to display a bimodal
distribution of membrane potentials. We measured the
membrane potentials of control cortical astrocytes (n 5
113) and confirmed this bimodal distribution (Fig. 2A).
The distribution was well fit by a double Gaussian func-
tion with peaks at about 268 mV and 241 mV, with a dis-
tribution of 77% in the predominant hyperpolarized popu-
lation. After knock-down of Kir4.1 channel subunits, the
distribution of membrane potentials (n 5 74) was notably
altered and could no longer be fit by a double Gaussian
function. The hyperpolarized cells were mostly absent and
the distribution was fit by a single Gaussian function with
a peak at 245 mV (Fig. 2B).
Using a voltage-step and ramp protocol (Fig. 3), the cur-
rent responses of Kir4.1 siRNA transfected and control cells
were determined 3–5 days after transfection. Representa-
tive current traces from a control and a siRNA transfected
astrocyte are superimposed in Fig. 3A. Upon opening of the
patch, these two particular cells had comparable membrane
(‘‘entry’’) potentials of about 250 mV, and were held at this
potential for the recording. There was a strong reduction in
the inward currents in the cell treated with Kir4.1 siRNA as
compared with the control. A summary of the inward and
outward current densities in response to voltage is shown in
Fig. 3B. While there is a marked reduction of the inward
current density in Kir4.1 siRNA astrocytes, there is little
difference in the outward current density of these cells.
with HiPerfect and no siRNA) and Kir4.1 siRNA at two concentrations 5
nM and 10 nM. Cells were harvested for Western Blot 4 days after trans-
fection. B: Quantification of the decrease in Kir4.1 protein 3–4 days after
Kir4.1 siRNA transfection (10 nM; n 5 5). Intensity of the chemilumi-
nescence signal was corrected for minor changes in protein content after
densitometry analysis of the India ink stained membrane.
A: Western Blot for control, mock transfection (transfected
siRNA transfected astrocytes. A: Control (mock-transfected) astrocytes
B: Astrocytes transfected with Kir4.1 siRNA (10 nM) 3–5 days prior to
Distribution of the membrane potentials of control and Kir4.1
Kir4.1 AND ASTROCYTIC K+AND GLUTAMATE BUFFERING
GLIA DOI 10.1002/glia
Inward current densities (pA/pF) measured at 100 mV neg-
ative to the holding potentials were significantly different
(P < 0.01; two-tailed t-test) for control and Kir4.1 siRNA
treated astrocytes (29.9 6 1.9 pA/pF and 23.2 6 0.4 pA/pF,
These results were supported by measuring the K1-cur-
rents in response to different [K1]olevels in control and
Kir4.1-suppressed astrocytes. The astrocytes were clamped
at a holding potential equal to their membrane potential in
a solution containing 3 mM [K1]oand this solution was
changed to 10 or 30 mM [K1]o. Inward currents produced
by 10 and 30 mM [K1]oin control astrocytes were 309.9 6
104.2 pA and 1357.8 6 514.7 pA, respectively (n 5 23). Cur-
rents in Kir4.1 siRNA treated cells (n 5 29) were 46.4 6
14.6 pA in 10 mM [K1]oand 190.5 6 44.3 in 30 mM [K1]o.
This was a depression of inward current by an average of
85% in both cases.
These effects appear to be due to specific knock-down
of Kir4.1 protein as there was no difference in (i) mem-
brane potential or (ii) currents in response to changes in
[K1]o between control astrocytes and cells transfected
with 20 nM of nonsilencing RNA (Ambion, Austin, TX)
(membrane potential 264.2 6 5.2 vs. 259.8 6 3.4 mV and
response to K1step from 3 to 30 mM 2198.3 6 67.8 vs.
2203.1 6 45.3 pA for control (n 5 9) vs. nonsilencing (n 5
14) cells, respectively).
It is noteworthy to point out that the observed reduction
in current responses to K1steps for Kir4.1-suppressed
astrocytes was not simply due to a depolarizing shift in
the membrane potential. If we include only astrocytes
(control and transfected) with membrane (‘‘entry’’) poten-
tials between 240 and 260 mV, the response to elevated
[K1]o(30 mM) was 8-fold less in transfected cells than in
control cells (2802.5 6 435.8 pA; n 5 8 for control vs.
2108.7 6 25.2 for Kir4.1 siRNA transfected; n 5 16; P <
0.05 for a two-tailed t-test). In this series, the mean mem-
brane potentials were not significantly different between
the two groups (250.5 6 2.6 mV and 246.0 6 1.4 mV for
control and transfected cells, respectively).
In a final series of experiments, we assessed the gluta-
mate uptake capability of astrocytes. The ability of astro-
cytes to clear glutamate in the presence and absence of
Ba21(1, 10, and 100 lM) was determined. Sixty minutes
after incubation with 300 lL of 400 lM glutamate (0 Ba21)
in serum-free media, the glutamate concentrations in the
media were reduced by 33.1% (Fig. 4A). There was a 30.5%
reduction of glutamate clearance when Kir channels were
blocked by 100 lM Ba21(from 132.4 lM glutamate cleared
in control cells to only 92.0 lM glutamate cleared in the
presence of barium; P < 0.01 one-way ANOVA followed by
Knock-down of Kir4.1 protein also reduced the gluta-
mate uptake by astrocytes. There was no difference in glu-
tamate clearance between control (untransfected) cells
and mock-transfected cells but transfection with Kir4.1
siRNA significantly decreased the glutamate clearance by
57.0% as compared with clearance by mock-transfected
astrocytes (P < 0.01 one-way ANOVA followed by Tukey’s
test) (Fig. 4B).
Maintenance of a Hyperpolarized
In this study, we used RNA interference to test the puta-
tive role(s) of Kir4.1 (-containing) channels in the mainte-
nance of the membrane potential, the mediation of trans-
membrane K1currents, and glutamate uptake of cultured
cortical astrocytes. We show that indeed, Kir4.1 is a crucial
player in all three functions.
In control astrocytes, membrane potential distribution
was clearly bi-modal, as reported previously (McKhann
et al., 1997; Zhou and Kimelberg, 2000) with the majority
of cells being strongly hyperpolarized. After depletion of
Kir4.1, only a depolarized population of cells remained.
Together with the finding that only about 50% of brain
cortical astrocytes. A: Representative current recordings of control and
Kir4.1 siRNA-treated (3 days) astrocytes. Currents are shown in response to
steps to 2100 mV, then a ramp to 1100 mV. Vh5 Vm, where the cells demon-
strate zero membrane current. B: Current density (pA/pF 6 s.e.m.) of control
and Kir4.1 siRNA-treated astrocytes in response to voltage steps from 2100
to 180 mV from the equilibrium potential (marked as 0 on the voltage scale).
Data were collected from 10 control and 14 siRNA transfected cells.
Suppression of Kir4.1 markedly reduces the inward current in
KUCHERYAVYKH ET AL.
GLIA DOI 10.1002/glia
astrocytes express Kir4.1 (Higashi et al., 2001; Schr€ oder
et al., 2002), this suggests that Kir4.1 is necessary for
strong hyperpolarization of the membrane potential in
these cortical astrocytes. Consistently, a role in setting
the hyperpolarized membrane potential was recently
described for Kir4.1 in astrocytes of a respiratory control
center in brain (Neusch et al., 2006) and in spinal cord
astrocytes (Olsen et al., 2006). It remains to be clarified
whether homomeric Kir4.1 channels or heteromeric Kir
channels containing the Kir4.1 subunit are involved.
Both homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1
channels have been identified in astrocytes of mouse cor-
tex using co-immunoprecipitation (Hibino et al., 2004).
However, our data also underline that Kir4.1-containing
channels are not the only channels contributing to the
overall negative membrane potential of astrocytes. After
knock-down of Kir4.1 (-containing) channels in our cells,
inward currents were dramatically reduced whereas ro-
bust outward currents remained under physiological K1
conditions (Fig. 3). This is compatible with the presence of
2P-domain K1channels (for review see Goldstein et al.,
2001) or Kv channels (Bekar et al., 2005). Gnatenco et al.
(2002) have shown that mRNA for TASK-1, TASK-3, and
TREK-2 was present in cultured cortical astrocytes. Out-
wardly rectifying K1channels have been ascribed in role
in maintaining an ‘‘auxiliary,’’ depolarized membrane
potential in M€ uller cells (Pannicke et al., 2000); these may
be 2P-domain K1channels (Skatchkov et al., 2006a).
K1Buffering Currents Through
In addition to their role in maintaining the hyperpolar-
ized membrane potential of (at least, some) glial cells
(Kofuji et al., 2000), Kir4.1 channels are believed to be
involved in a specialized K1clearance mechanism called
potassium buffering (Orkand et al., 1966) or potassium
siphoning (Newman, 1984). This mechanism involves (i)
K1influx into glial cells at sites of high [K1]o, (ii) spatial
dissipation of K1within the glial cytoplasm or—via gap
junctions—within the glial network, and finally (iii) dis-
posal of K1via astrocytic endfeet into capillaries (or the
vitreous body in the case of the retina 5 potassium
siphoning). It relies on a high, locally distinct potassium
permeability of the glial cell membrane (for review see
Skatchkov et al., 2006b).
We assessed the potential ability of cells to buffer potas-
sium by measuring membrane currents in response to ex-
tracellular K1steps (Skatchkov et al., 1999; Zhou and
Kimelberg, 2000). In astrocytes with Kir4.1 subunit knock-
down, we found an 85% reduction of inward currents. This
implies that Kir4.1 (-containing) channels may carry the
vast majority of spatial buffering currents of cortical astro-
cytes in vivo, and at both K1influx (perisynaptic sheaths)
and efflux sites (endfeet). Indeed, Kir4.1 immunoreactivity
was demonstrated at the light- and electron microscopical
et al., 2001; Poopalasundaram et al., 2000). The observed
reductioninoutwardcurrent in ourstudyis consistent with
the finding of D’Ambrosio et al. (2002) indicating that Kir
The K1siphoning process in the cortex may involve dif-
ferent mediators than in the retina where it has been sug-
gested that Kir2.1 channels, in retinal M€ uller (glial) cells,
may be responsible for K1influx within the synaptic
layers, whereas Kir4.1 channels may be mainly responsi-
ble for K1release into the vitreous and the blood vessels
(Kofuji and Newman, 2004; Kofuji et al., 2002). Expres-
sion of functional Kir2-type channel subunits in astrocytes
remains controversial (Jansen et al., 2005; Pruss et al.,
2005; Schr€ oder et al., 2002; Stonehouse et al., 1999).
However, in the present study, we demonstrate an almost
Cortical Astrocytes. A: Dose response effects of barium (0, 1, 10, and
100 lM) on glutamate clearance by astrocytes. The concentration of glu-
tamate was determined 60 min after addition of 400 lM glutamate in
media. * indicates significant difference from 400 lM glutamate with
Effect of Ba21and Kir4.1 siRNA on Glutamate Clearance by0 Ba21(P < 0.01) with n 5 7–9 per group. B: Glutamate clearance by
control, mock-transfected and Kir4.1 siRNA (5 nM)-transfected astro-
cytes. * indicates significant difference from control (untransfected) and
mock-transfected (P < 0.01) with n 5 9–10 per group.
Kir4.1 AND ASTROCYTIC K+AND GLUTAMATE BUFFERING
GLIA DOI 10.1002/glia
complete loss of inward currents when Kir4.1 is depressed
(Fig. 3); this is not indicative of any substantial functional
expression of other Kir (such as Kir2) channels.
Glutamate is the major excitatory neurotransmitter in
the mammalian central nervous system (Erecinska and
Silver, 1990; Fonnum, 1984). Rapid removal of glutamate
from the extracellular space is essential both for survival
and normal functioning of neurons. Although glutamate
transporters are expressed in both astrocytes and neu-
rons, astrocytes are the cell type primarily responsible
for glutamate uptake (Rothstein et al., 1996; Tanaka
et al., 1997). Glutamate clearance depends upon a hyper-
polarized membrane potential of the glial cells (Barbour
et al., 1991; Brew and Attwell, 1987). In the present
study, we found that blockade or suppression of Kir4.1
subunits substantially inhibited the ability of astrocytes
to clear extracellular glutamate. This reduced glutamate
uptake may be due to overall depolarization of astrocytes
after suppression of Kir4.1-containing channels. Gluta-
mate transport is a multistep process beginning with
binding of substrate and ions, followed by translocation
and unbinding of substrate and ions. Depolarization has
been shown to inhibit the translocation step of this pro-
cess (not the affinity of glutamate for the transporter)
(Mennerick et al., 1999; Otis and Kavanaugh, 2000).
Potential In Vivo Consequences of Reduced
Cortical Astrocyte Kir Channels
There are many potential consequences of a dysfunction
or lack of Kir4.1 (-containing) channels within the brain.
The most obvious is that elevations in potassium due to
decreased potassium buffering will lead to hyperexcitabil-
ity of neurons and, ultimately, to seizures. Indeed, Jansen
et al. (2005) have recently demonstrated alterations of Kir
channels in astrocytes associated with epileptogenesis in
a mouse model of the tuberous sclerosis complex. Further-
more, mislocalization of aquaporin-4 which is normally co-
localized with Kir4.1 protein (Nagelhus et al., 1999, 2004)
results in delayed K1clearance and increased seizure ac-
tivity in mice (Amiry-Moghaddam et al., 2003). In addi-
tion, the depolarized membrane potential of Kir4.1-defi-
cient astrocytes will impair the function of electrogenic
transporters, such as the glutamate transporter (Brew
and Attwell, 1987). This would result in decreased gluta-
mate clearance and increased extracellular glutamate,
which eventually must aggravate the neuronal hyperex-
citability and seizure activity, and ultimately may cause
excitotoxic neuronal cell death.
The authors thank Paola L? opez Pieraldi, Natalia Skach-
kova, Martha Ricarte, Maria Gonzalez, and Noel Mayol
for their reliable technical assistance and Dr. Thomas
Pannicke for discussion of these experiments.
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Kir4.1 AND ASTROCYTIC K+AND GLUTAMATE BUFFERING
GLIA DOI 10.1002/glia