Two pore domain potassium channels in cerebral ischemia: a focus on K2P9.1 (TASK3, KCNK9)

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 parameters 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 Ca 2+activated K + channels and ATP-sensitive K + channels [2,3]) might be neuroprotective as they counterbalance a prolonged harmful influx of Ca 2+ ions via different pathways including a reversal of the Na + /Ca 2+ antiporter and voltage-dependent Ca 2+ channels. Furthermore, an enhancement of the Mg 2+ block of NMDA receptors (Nmethyl D-aspartate) in postsynaptic neurons [4] is thought to protect against glutamate excitotoxicity [5,6].
Concerning the recently identified family of two-pore domain potassium channels (K 2P channels), several members have been shown to play a major role in critical conditions leading to cerebral ischemia. K 2P 2.1 -/mice displayed significantly less neuronal survival rates in a model of cerebral ischemia [7]. These data were confirmed by the neuroprotective effect of several K 2P 2.1 channel activators (e.g. alpha linelonic acid or riluzole [8][9][10]). On the other hand, genetic depletion of another family member, namely K 2P 3.1, 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 K 2P 9.1 (TASK3; KCNK9) in a tMCAO model using previously described K 2P 9.1 -/mice [13].

Electrophysiology
Slices were transferred in a recording chamber and thalamic neurons of the dLGN were visualized with a microscope 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 solution containing (in mM): K-gluconate, 88; K 3 -citrate, 20; NaCl, 10; HEPES, 10; MgCl 2 , 1; CaCl 2 , 0.5; BAPTA, 3; phosphocreatin, 15; Mg-ATP, 3; Na-GTP, 0.5. The internal 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 2.5 mM, NaH 2 PO 4 1.25 mM, HEPES 30 mM, MgSO 4 2 mM, CaCl 2 2 mM and dextrose 10 mM. Whole-cell patch-clamp recordings were measured from relay neu-rons of the dLGN with an EPC-10 amplifier (HEKA Elektronik, 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 solution containing 0 mM Mg 2+ and 0 mM Ca 2+ ; the osmolality 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 K 2P 9.1 -/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% isoflurane (Abbott, Wiesbaden, Germany) in a 70% N 2 O/30% O 2 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 external 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 reperfusion. 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, Axminster, United Kingdom) was used to monitor cerebral blood flow [22] in wildtype, K 2P 9.1 -/and sham-treated animals (n = 4/group) before surgery (baseline), immediately after MCA occlusion, and 5 minutes after removal of the occluding monofilament (reperfusion). Cerebral perfusion did not differ between the groups at any time point (Additional File 1, Figure S1).

Determination of infarct size
Mice were sacrificed 24 hours after tMCAO, respectively. Brains were quickly removed and cut in 2 mm thick coronal 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 software, 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 Oneway 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 K 2P 9.1 channels K 2P 9.1-like currents have been demonstrated in a number of different central nervous system neurons [14,[26][27][28]. As highly specific inhibitors for K 2P channel subtypes are not available, different semi-selective blockers and experimental strategies to distinguish between these channels were established. Among them, extracellular reduction of divalent cations was introduced to increase potassium outward currents through K 2P 9.1 channels [17]. Currentvoltage relationships (I/V) of the standing outward current of wildtype and K 2P 9.1 -/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 (I SO ) of 322.33 ± 30.20 pA was measured at -35 mV (Fig. 1A). Application of hyperpolarizing voltage ramps induced a complex current response. The wave form of this response was indicative for the contribution of current through outwardly rectifying TASK channels as well as inwardly rectifying K + channels (Fig. 1A, black trace). Removal of extracellular divalent cations resulted in a significant increase of I SO 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 cationfree conditions was calculated by numerical subtraction of control currents from currents recorded under cationfree conditions [14]. The I/V relationship of the cationsensitive current was typical of TASK channels with a strong outward rectification and a reversal potential close to the expected potassium equilibrium potential ( Fig. 1B; E K = -104 mV). These findings indicate a strong contribution of K 2P 9.1 channels to the I SO of thalamocortical (TC) neurons in wildtype mice.

Neurons from K 2P 9.1 -/and wildtype animals show no significant differences upon extracellular acidification
Sensitivity to extracellular acidification is a hallmark of TASK channels and a reduction of the extracellular pH value can be typically observed under ischemic conditions. 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 I SO amplitudes in both genotypes (Fig. 2A). The degree of I SO reduction was not different in wildtype (68.31 ± 9.80%) and K 2P 9.1 -/neurons (69.92 ± 11.65%; n = 5; p = 0.91; Fig. 2B).

Discussion
The results of the present study can be summarized as follows: (1) A pH-and divalent cation-sensitive I SO is present in TC neurons of the dLGN. (2) The divalent cation-sensitive component is characterized by outward rectification and a reversal potential close to the potassium equilibrium potential. (3) The I SO of neurons recorded from brain slices of K 2P 9.1 -/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, K 2P 9.1 -/animals showed a tendency to reduced  infarct volumes 24 hours after undergoing 60 min of tMCAO compared to wildtype controls although these results were not statistically significant. (5) It is concluded that K 2P 9.1-containing homodimeric and heterodimeric channels significantly contribute to I SO in TC neurons from wildtype mice and that K 2P 9.1 channels have only a minor impact on infarct volume and motor function following tMCAO compared to other members of the K 2P channel family.

Contribution of TASK channel subtypes to I SO in TC neurons
During development, the mouse thalamus is characterized by high K 2P 3.1 gene expression at P0 and displays moderate expression levels throughout postnatal stages [31]. K 2P 9.1 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-nels can be K 2P 3.1 homodimers, K 2P 9.1 homodimers, and K 2P 3.1/K 2P 9.1 heterodimers [32][33][34][35]. Although K 2P 3.1 and K 2P 9.1 show high sequence homology, they differ in their sensitivity to extracellular divalent cations (Mg 2+ , Ca 2+ ) based on the presence of a glutamate residue at position 70 in K 2P 9.1 channels [17]. While the conductance of K 2P 3.1 homodimeric channels is unaffected, the conductance of K 2P 9.1 homodimeric and K 2P 3.1/K 2P 9.1 heterodimeric channels is strongly reduced in the presence of divalent cations [17,33]. Therefore the increase in I SO following removal of extracellular divalent cations which was found in cells from different rodent strain (Long Evans rats, wildtype mice, K 2P 3.1 -/mice) point to the functional expression of K 2P 9.1 homodimeric and K 2P 3.1/ K 2P 9.1 heterodimeric channels in TC neurons.   Homodimeric and heterodimeric TASK channels also differ in their pH-sensitivity. While K 2P 3.1/K 2P 9.1 channel constructs have a pH-sensitivity (pK approximately 7.3) in the physiological range which is closer to that of K 2P 3.1 channels (pK approximately 7.5) than K 2P 9.1 channels (pK approximately 6.8) [34]. In the present study no significant difference was found for the decrease in I SO amplitude when the pH was shifted to a value of 6.0. Therefore the pH-and divalent cation-sensitivities of native TASK-like currents in TC neurons is best represented by K 2P 3.1/K 2P 9.1 heterodimeric channels. However, additional modulators (isoflurane, Zn 2+ , ruthenium red) have to be tested to get more indications for the ratio of homodimeric to heterodimeric TASK channels in these neurons.