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http://www.etsmjournal.com/content/2/1/14Open AccessR E S E A R C H
ResearchTwo pore domain potassium channels in cerebral
ischemia: a focus on K2P9.1 (TASK3, KCNK9)
Petra Ehling†1, Stefan Bittner†2, Nicole Bobak3, Tobias Schwarz2, Heinz Wiendl3, Thomas Budde1,
Christoph Kleinschnitz*†2 and Sven G Meuth*†2,3
Abstract
Background: Recently, members of the two-pore domain potassium channel family (K2P channels) could be shown to
be involved in mechanisms contributing to neuronal damage after cerebral ischemia. K2P3.1-/- animals showed larger
infarct volumes and a worse functional outcome following experimentally induced ischemic stroke. Here, we question
the role of the closely related K2P channel K2P9.1.
Methods: We combine electrophysiological recordings in brain-slice preparations of wildtype and K2P9.1-/- mice with
an in vivo model of cerebral ischemia (transient middle cerebral artery occlusion (tMCAO)) to depict a functional
impact of K2P9.1 in stroke formation.
Results: Patch-clamp recordings reveal that currents mediated through K2P9.1 can be obtained in slice preparations of
the dorsal lateral geniculate nucleus (dLGN) as a model of central nervous relay neurons. Current characteristics are
indicative of K2P9.1 as they display an increase upon removal of extracellular divalent cations, an outward rectification
and a reversal potential close to the potassium equilibrium potential. Lowering extracellular pH values from 7.35 to 6.0
showed comparable current reductions in neurons from wildtype and K2P9.1-/- mice (68.31 ± 9.80% and 69.92 ± 11.65%,
respectively). These results could be translated in an in vivo model of cerebral ischemia where infarct volumes and
functional outcomes showed a none significant tendency towards smaller infarct volumes in K2P9.1-/- animals
compared to wildtype mice 24 hours after 60 min of tMCAO induction (60.50 ± 17.31 mm3 and 47.10 ± 19.26 mm3,
respectively).
Conclusions: Together with findings from earlier studies on K2P2.1-/- and K2P3.1-/- mice, the results of the present study
on K2P9.1-/- mice indicate a differential contribution of K2P channel subtypes to the diverse and complex in vivo effects
in rodent models of cerebral ischemia.
Background
Although ischemic stroke represents a major health care
problem with a high rate of permanent disability or even
death, the underlying molecular mechanisms leading to
neuronal death are still poorly understood [1]. However,
ion channels which can influence basal cellular parame-
ters are thought to play a major role within this context.
Activation of potassium channels results in membrane
hyperpolarization thereby decreasing neuronal activity
and cell death under pathophysiological conditions.
Additionally, K+ channels (e.g. large conductance Ca2+-
activated K+ channels and ATP-sensitive K+ channels
[2,3]) might be neuroprotective as they counterbalance a
prolonged harmful influx of Ca2+ ions via different path-
ways including a reversal of the Na+/Ca2+ antiporter and
voltage-dependent Ca2+ channels. Furthermore, an
enhancement of the Mg2+ block of NMDA receptors (N-
methyl D-aspartate) in postsynaptic neurons [4] is
thought to protect against glutamate excitotoxicity [5,6].
* Correspondence: christoph.kleinschnitz@mail.uni-wuerzburg.de, sven.meuth@gmx.de
2 University of Wuerzburg, Department of Neurology, Josef-Schneider Str. 11,
97080 Wuerzburg, Germany
3 University of Muenster, Neurological Clinic - Inflammatory Disorders of the
Central Nerveous System and Neurooncology, Mendelstr. 7, 48149 Muenster, © 2010 Ehling et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Germany
† Contributed equally
Full list of author information is available at the end of the article
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Concerning the recently identified family of two-pore
domain potassium channels (K2P channels), several mem-
bers have been shown to play a major role in critical con-
ditions leading to cerebral ischemia. K2P2.1-/- mice
displayed significantly less neuronal survival rates in a
model of cerebral ischemia [7]. These data were con-
firmed by the neuroprotective effect of several K2P2.1
channel activators (e.g. alpha linelonic acid or riluzole [8-
10]). On the other hand, genetic depletion of another
family member, namely K2P3.1, resulted in increased
infarct volumes following transient or permanent middle
cerebral artery occlusion (MCAO) [11,12]. Based on
sequence homologies and similar biophysical properties,
it was suggested that related channel family members
might also be of importance under these circumstances.
We challenged the role of K2P9.1 (TASK3; KCNK9) in a
tMCAO model using previously described K2P9.1-/- mice
[13].
Methods
Slice preparation
Thalamic tissue slices including the dorsal lateral genicu-
late nucleus (dLGN) were prepared from 14 - 22 days old
male C57BL/6 or K2P9.1-/- mice [13] as described earlier
[14]. Coronal sections were cut on a vibratome
(Vibratome®, Series 1000 Classic, St. Louis, USA) in an
ice-chilled solution containing (mM): Sucrose, 200;
PIPES, 20; KCl, 2.5; NaH2PO4, 1.25; MgSO4, 10; CaCl2,
0.5; dextrose, 10; pH 7.35 adjusted with NaOH. Prior to
the recording procedure, slices were kept submerged in
artificial cerebrospinal fluid (ACSF, mM): NaCl, 125; KCl,
2.5; NaH2PO4, 1.25; NaHCO3, 24; MgSO4, 2; CaCl2, 2;
dextrose, 10; pH adjusted to 7.35 by bubbling with a mix-
ture of 95% O2 and 5% CO2.
Electrophysiology
Slices were transferred in a recording chamber and thal-
amic neurons of the dLGN were visualized with a micro-
scope equipped with infrared-differential interference
contrast optics [15]. Whole-cell recording pipettes were
fabricated from borosilicate glass (GT150T-10, Clark
Electromedical Instruments, Pangbourne, UK; typical
resistance 2-3 MΩ) and filled with an intracellular solu-
tion containing (in mM): K-gluconate, 88; K3-citrate, 20;
NaCl, 10; HEPES, 10; MgCl2, 1; CaCl2, 0.5; BAPTA, 3;
phosphocreatin, 15; Mg-ATP, 3; Na-GTP, 0.5. The inter-
nal solution was set to a pH value of 7.25 using KOH and
an osmolarity of 295 mOsm/kg. Slices were continuously
superfused with a solution containing NaCl 125 mM, KCl
rons of the dLGN with an EPC-10 amplifier (HEKA Elek-
tronik, Lamprecht, Germany) and digitally analyzed
using Pulse software (HEKA Elektronik; [16]). pH was
adjusted to 7.35 or 6.0 with HCl. For divalent-cation-free
conditions we switched from control solution to a solu-
tion containing 0 mM Mg2+ and 0 mM Ca2+; the osmolal-
ity was kept constant at 305 mosmol kg-1 by adding 4 mM
NaCl [17]. All cells had a resting membrane potential
negative to -65 mV, the access resistance was in the range
of 5-15 MΩ and series resistance compensation of more
than 40% was routinely used.
Induction of cerebral ischemia
Animal experiments were approved by governmental
agencies for animal research and conducted according to
the recommendations for research in mechanism-driven
basic stroke studies [18]. Focal cerebral ischemia was
induced in 6-8 weeks old male C57BL/6 and K2P9.1-/-
mice [13] weighing 20-25 g by transient middle cerebral
artery occlusion (tMCAO) as described previously
[19,20]. Briefly, mice were anesthetized with 2.5% isoflu-
rane (Abbott, Wiesbaden, Germany) in a 70% N2O/30%
O2 mixture. Core body temperature was maintained at
37°C throughout surgery using a feedback-controlled
heating device. Following a midline skin incision in the
neck, the proximal common carotid artery and the exter-
nal carotid artery were ligated and a standardized silicon
rubber-coated 6.0 nylon monofilament (6021; Doccol
Corp., CA, USA) was inserted and advanced via the right
internal carotid artery to occlude the origin of the right
MCA. The intraluminal suture was left in situ for 1 hour,
respectively. Then animals were re-anesthetized and the
occluding monofilament was withdrawn to allow reperfu-
sion. After 24 hours neurological deficits were scored by
two blinded investigators and quantified according to
Bederson [21]: 0, no deficit; 1, forelimb flexion; 2, as for 1,
plus decreased resistance to lateral push; 3, unidirectional
circling; 4, longitudinal spinning; 5, no movement. For
the gript test, the mouse was placed midway on a string
between two supports and rated as follows: 0, falls off; 1,
hangs onto string by one or both forepaws; 2, as for 1, and
attempts to climb onto string; 3, hangs onto string by one
or both forepaws plus one or both hindpaws; 4, hangs
onto string by fore- and hindpaws plus tail wrapped
around string; 5, escape (to the supports).
Laser doppler flowmetry (Moor Instruments, Axmin-
ster, United Kingdom) was used to monitor cerebral
blood flow [22] in wildtype, K2P9.1-/- and sham-treated
animals (n = 4/group) before surgery (baseline), immedi-
ately after MCA occlusion, and 5 minutes after removal2.5 mM, NaH2PO4 1.25 mM, HEPES 30 mM, MgSO4 2
mM, CaCl2 2 mM and dextrose 10 mM. Whole-cell
patch-clamp recordings were measured from relay neu-
of the occluding monofilament (reperfusion). Cerebral
perfusion did not differ between the groups at any time
point (Additional File 1, Figure S1).
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Determination of infarct size
Mice were sacrificed 24 hours after tMCAO, respectively.
Brains were quickly removed and cut in 2 mm thick coro-
nal sections using a mouse brain slice matrix. The slices
were stained with 2% 2,3,5-triphenyltetrazolium chloride
(TTC; Sigma-Aldrich, St. Louis, MO) in PBS to visualize
the infarctions. Planimetric measurements (ImageJ soft-
ware, National Institutes of Health, Bethesda, MD)
blinded to the treatment groups were used to calculate
lesion volumes, which were corrected for brain edema as
described [23,24].
Statistical analysis
Electrophysiological data and results from the animal
experiments were analyzed by a modified student's t test
for small samples [25] or by a Bonferroni-corrected One-
way ANOVA in case of multiple comparisons using
PrismGraph 4.0 software (GraphPad Software, San
Diego, CA) or Origin® (Microcal). P-values < 0.05 were
considered statistically significant.
Results
Thalamic relay neurons as a model system of central
nervous system neurons display electrophysiological
properties indicative of currents through K2P9.1 channels
K2P9.1-like currents have been demonstrated in a number
of different central nervous system neurons [14,26-28].
As highly specific inhibitors for K2P channel subtypes are
not available, different semi-selective blockers and exper-
imental strategies to distinguish between these channels
were established. Among them, extracellular reduction of
divalent cations was introduced to increase potassium
outward currents through K2P9.1 channels [17]. Current-
voltage relationships (I/V) of the standing outward cur-
rent of wildtype and K2P9.1-/- mice were investigated by
ramping the membrane potential from -35 mV to -125
mV over 800 ms (Fig. 1A, inset; [29,30]). Under control
conditions a standing outward current (ISO) of 322.33 ±
30.20 pA was measured at -35 mV (Fig. 1A). Application
of hyperpolarizing voltage ramps induced a complex cur-
rent response. The wave form of this response was indic-
ative for the contribution of current through outwardly
rectifying TASK channels as well as inwardly rectifying
K+ channels (Fig. 1A, black trace). Removal of extracellu-
lar divalent cations resulted in a significant increase of ISO
by 35.47 ± 9.59% compared to control conditions (n = 6, p
= 0.007; Fig. 1A). Ramp responses revealed a clear
increase in the outwardly rectifying component (Fig. 1A,
gray trace). The current sensitive to administering cation-
free conditions was calculated by numerical subtraction
to the expected potassium equilibrium potential (Fig. 1B;
EK = -104 mV). These findings indicate a strong contribu-
tion of K2P9.1 channels to the ISO of thalamocortical (TC)
neurons in wildtype mice.
Neurons from K2P9.1-/- and wildtype animals show no
Figure 1 Whole cell currents recorded from relay neurons in thal-
amic slice preparations show characteristics indicative for K2P9.1
channels. (A) Mean ramp currents under control recording conditions
(ctrl) and after removal of extracellular divalent cations (div. cation
free). Inset: Currents were evoked by ramping the membrane potential
from -35 mV to -125 mV over 800 ms. (B) I-V relationship of the divalent
cation-sensitive current component shows characteristics indicative of
TASK channels. I = current; ISO = standing outward current; VM = mem-
brane potential.
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Vm [mV]of control currents from currents recorded under cation-
free conditions [14]. The I/V relationship of the cation-
sensitive current was typical of TASK channels with a
strong outward rectification and a reversal potential close
significant differences upon extracellular acidification
Sensitivity to extracellular acidification is a hallmark of
TASK channels and a reduction of the extracellular pH
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value can be typically observed under ischemic condi-
tions. In a next experimental step we therefore mimicked
cerebral ischemia by lowering the extracellular pH from
control conditions (7.35) to 6.0. This maneuver resulted
in a significant (p < 0.05) reduction of ISO amplitudes in
both genotypes (Fig. 2A). The degree of ISO reduction was
not different in wildtype (68.31 ± 9.80%) and K2P9.1-/-
neurons (69.92 ± 11.65%; n = 5; p = 0.91; Fig. 2B).
Genetic ablation of K2P9.1 channels trends to result in a not
significant reduction of stroke development after tMCAO
Stroke volumes of wildtype and K2P9.1-/- mice were deter-
mined 24 hours after animals subjected to 60 min of
tMCAO. Wildtype animals showed stroke volumes of
60.50 ± 17.31 mm3 while K2P9.1-/- mice displayed infarct
areas of 47.10 ± 19.26 mm3 (n = 10 and 8; p = 0.23; Fig. 3A
and 3B). In accordance with this tendency towards none
significantly smaller infarct sizes in K2P9.1-/-, no function-
ally relevant differences could be found for the Bederson
score (WT: 1.83 ± 0.98; K2P9.1-/-: 2.14 ± 0.80; n = 6; p =
0.55; Fig. 3C) and the grip test (WT: 3.17 ± 1.13; K2P9.1-/-:
4.29 ± 0.64; n = 6; p = 0.09; Fig. 3D).
Discussion
The results of the present study can be summarized as
follows: (1) A pH- and divalent cation-sensitive ISO is
present in TC neurons of the dLGN. (2) The divalent cat-
ion-sensitive component is characterized by outward rec-
tification and a reversal potential close to the potassium
equilibrium potential. (3) The ISO of neurons recorded
from brain slices of K2P9.1-/- mice and wildtype mice
showed comparable pH-sensitivity during extracellular
pH changes from 7.35 to 6.0. (4) In a model of cerebral
ischemia, K2P9.1-/- animals showed a tendency to reduced
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Figure 2 pH dependence of the standing outward currents in K2P9.1-/- neurons shows no difference compared to wildtype mice (pH 7.35 T
pH 6.0). (A) Ramp currents under control conditions (pH 7.35) and after extracellular acidification (pH 6.0) in wildtype (WT; left panel) and K2P9.1-/- mice
(right panel). (B) Bar graph representation of current reduction after extracellular pH lowering in wildtype (WT) and K2P9.1-/- mice.
Page 5
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infarct volumes 24 hours after undergoing 60 min of
tMCAO compared to wildtype controls although these
results were not statistically significant. (5) It is con-
cluded that K2P9.1-containing homodimeric and het-
erodimeric channels significantly contribute to ISO in TC
neurons from wildtype mice and that K2P9.1 channels
have only a minor impact on infarct volume and motor
function following tMCAO compared to other members
of the K2P channel family.
Contribution of TASK channel subtypes to ISO in TC neurons
During development, the mouse thalamus is character-
ized by high K2P3.1 gene expression at P0 and displays
moderate expression levels throughout postnatal stages
nels can be K2P3.1 homodimers, K2P9.1 homodimers, and
K2P3.1/K2P9.1 heterodimers [32-35]. Although K2P3.1 and
K2P9.1 show high sequence homology, they differ in their
sensitivity to extracellular divalent cations (Mg2+, Ca2+)
based on the presence of a glutamate residue at position
70 in K2P9.1 channels [17]. While the conductance of
K2P3.1 homodimeric channels is unaffected, the conduc-
tance of K2P9.1 homodimeric and K2P3.1/K2P9.1 heterodi-
meric channels is strongly reduced in the presence of
divalent cations [17,33]. Therefore the increase in ISO fol-
lowing removal of extracellular divalent cations which
was found in cells from different rodent strain (Long
Evans rats, wildtype mice, K2P3.1-/- mice) point to the
functional expression of K2P9.1 homodimeric and K2P3.1/
Figure 3 Infarct volumes 24 h after 60 min MCA occlusion in wildtype and K2P9.1-/- mice. (A) Representative TTC-stained images of three corre-
sponding coronal sections of control animals (WT) and K2P9.1-/- mice. (B) Mean brain infarct volumes calculated from (A) (control group: n = 10; K2P9.1-
/- mice: n = 8). (C) Mean Bederson score and (D) grip test from the animals shown in (B). ns = not significant.
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�&�[31]. K2P9.1 expression in many thalamic nuclei is rather
moderate for all developmental stages but is strong in
dLGN from P14 to adult stages. Functional TASK chan-
K2P9.1 heterodimeric channels in TC neurons.
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