KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

Abstract

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca²⁺ ]_i ) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca²⁺ ]_i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (K_Ca 2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of K_Ca 2.2 channels within 3 h after the onset of glutamate exposure. Activation of K_Ca 2 channels preserved K_Ca 2 expression and significantly reduced pathological increases in [Ca²⁺ ]_i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for K_Ca 2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.

Citation: Cell Death and Disease (2011) 2, e147; doi:10.1038/cddis.2011.30

& 2011 Macmillan Publishers Limited All rights reserved 2041-4889/11 http://www.nature.com/cddis

Web End =www.nature.com/cddis

KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

AM Dolga1, N Terpolilli2,3, F Kepura4, IM Nijholt5, H-G Knaus6, B DOrsi7, JHM Prehn7, ULM Eisel5, T Plant4, N Plesnila3,7

and C Culmsee*,1

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca2]i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca2]i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (KCa2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of KCa2.2 channels within 3 h after the onset of glutamate exposure. Activation of KCa2 channels preserved KCa2 expression and signicantly reduced pathological increases in [Ca2]i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for KCa2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.

Cell Death and Disease (2011) 2, e147; doi:http://dx.doi.org/10.1038/cddis.2011.30

Web End =10.1038/cddis.2011.30 ; published online 21 April 2011

Subject Category: Neuroscience

Excessive release of glutamate and uncontrolled neuronal excitations lead to progressive neuronal death following cerebral ischemia. In particular, glutamate-induced stimulation of a-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid and N-methyl-D-aspartate (NMDA) receptors mediates toxic increases in intracellular calcium concentrations ([Ca2]i). In

neurons, disrupted intracellular calcium homeostasis disturbs multiple metabolic processes thereby promoting progressive cell death.13 Consequently, inhibition of NMDA receptors has been extensively investigated in experimental and clinical stroke studies. For example, studies on NMDA receptor antagonists such as dizocilpine (MK-801) or memantine revealed neuroprotective effects against ischemia-induced neuronal death in vitro and ischemic brain damage in vivo,4,5

promoting disturbed [Ca2]i as a promising therapeutic target. However, inhibitors of NMDA receptors largely failed in clinical studies6 and, therefore, novel strategies controlling [Ca2]i homeostasis are warranted for the development of effective therapies of neurological disorders associated with excessive NMDA receptor stimulation.

It has been suggested that small-conductance calcium-activated potassium (KCNN/SK/KCa2) channels may control membrane excitability through attenuated NMDA receptor activity.7,8 In neurons, synaptic NMDA receptors are closely

associated with KCa2 channels in dendritic spines. Small increases in [Ca2]i lead to the activation of KCa2 channels, which induce afterhyperpolarization thereby providing a feedback control that prevents toxic increases in [Ca2]i

under physiological conditions.9

How KCa2 channel-dependent [Ca2]i regulation is altered under pathological conditions, in which glutamate toxicity has an essential role, is so far unknown. Thus, we address the question whether pharmacological modulation of KCa2 chan

nels attenuates glutamate-induced [Ca2]i deregulation and cell death in primary cultured neurons in vitro and reduces infarct size following cerebral ischemia in vivo.

Results

Activation of KCa2 channels prevents the increases in [Ca2]i after glutamate exposure in neurons. Glutamate immediately induced increases in uorescence intensities of the calcium-sensitive dye FURA-2 and these increases in [Ca2]i over control levels sustained until termination of the measurements (Figure 1a). These glutamate-induced alterations in [Ca2]i signals can be subdivided into three phases: an early phase was detected as a very fast and

1Institut fr Pharmakologie und Klinische Pharmazie, Fachbereich Pharmazie, Philipps-Universitat Marburg, Marburg, Germany; 2Walter Brendel Center for Experimental Medicine, Ludwig-Maximilians-Universitat Mnchen, Mnchen, Germany; 3Department of Neurosurgery, Ludwig-Maximilians-Universitat Mnchen, Mnchen, Germany; 4Pharmakologisches Institut, Fachbereich Medizin, Philipps-Universitat Marburg, Marburg, Germany; 5Department of Molecular Neurobiology, University of Groningen, Groningen, The Netherlands; 6Department for Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria and 7Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland*Corresponding author: C Culmsee, Institut fr Pharmakologie und Klinische Pharmazie, Fachbereich Pharmazie, Philipps-Universitat Marburg, Karl-von-Frisch-Strabe 1,

Marburg 35032, Germany. Tel: [phone]; Fax: [phone]; E-mail: mailto:[email protected]

Web End [email protected]

Keywords: calcium homeostasis; cerebral ischemia; KCa2 channels; glutamate excitotoxicity; neuroprotectionAbbreviations: NMDA, N-methyl-D-aspartate; [Ca2]i, intracellular cytosolic calcium concentration; KCa2, small-conductance calcium-activated potassium channels; IAHP, afterhyperpolarization current; DCD, delayed calcium deregulation; TTX, tetrodotoxi; MTT, (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; EGTA, ethylene glycol tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; BHQ, 2,5-di-t-butyl-1, 4-benzohydroquinone; ER, endoplasmatic reticulum; SERCA, sarco/ endoplasmic reticulum Ca2-ATPase; MCAo, middle cerebral artery occlusion; NF-kB, nuclear factor-k B; CAF, cytosine arabinoside

Received 24.9.10; revised 09.3.11; accepted 15.3.11; Edited by A Verkhrasky

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Figure 1 Effect of KCa2 channel modulators on the onset of glutamate-induced delayed calcium deregulation. (a) Neuronal cells were loaded with FURA-2 AM and then stimulated with glutamate (20 mM) in the presence or absence of NS309 (50 mM), NS8593 (50 mM) or apamin (1 mM) pre-treatment. Glutamate was applied to neurons after monitoring the cells for 1 min, as shown in the [Ca2]i kinetic prole. (a) Representative FURA-2 uorescence intensities from neurons stimulated with glutamate (at 1 min) in the presence of NS309, NS8593 or apamin. (bd) Depicted are representative traces of [Ca2]i FURA-ratios values (S.E.M.) stimulated with glutamate in the presence of the indicated KCa2 channel modulators (n 1525). (e) MTT analysis of primary neuronal cultures pre-treated with NS309 (50 mM) or apamin (10 mM) and challenged with glutamate (20 mM) for 24 h

(**Po0.01 versus glutamate-treated neurons, ANOVA, Scheffs test)

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pronounced increase in [Ca2]i followed by a second phase with a short decrease of [Ca2]i, and a third phase characterized by a sustained [Ca2]i increase that showed a higher amplitude than the early phase (Figure 1b). The length of the second phase varied in range from seconds to minutes in different experimental settings. Moreover, individual neurons show this intermediate decrease of [Ca2]i at different time intervals ranging from 3 to 4 min up to 10 min, which limits the detection of this proposed second phase in average Ca2 kinetics plotted from up to 25 neurons. The third phase of the Ca2 kinetic is regarded as delayed calcium deregulation (DCD), which contributes to delayed cellular death.1,10 DCD was detected for periods of up to 12 h (Supplementary Figure S1A). Measurements of [Ca2]i in cortical neurons did not show any spontaneous [Ca2]i spikes in both basal conditions or in glutamate-stimulated neurons. This was further demonstrated by application of tetrodotoxin (TTX), which is known to block the transient spontaneous [Ca2]i spikes. TTX failed to inuence DCD formation (Supplementary Figure S1B). This nding is consistent with other reports that founds that TTX does not block DCD formation1 and also does not prevent the delayed cell death.11

Single-cell uorescence imaging further revealed that pre-treatment of neurons with NS309, an activator of KCa2

channels,12 prevented increases in [Ca2]i, and signicantly reduced DCD (Figure 1). Whole-culture [Ca2]i recordings for more than 1 h conrmed that the KCa2 channel activator NS309 attenuated the glutamate-induced elevated levels of [Ca2]i similar to the observations obtained by single-cell imaging (Supplementary Figure S1A).

In contrast to the ndings with the KCa2 activator NS309, pharmacological inhibition of KCa2 channels with NS8593 further increased the glutamate-induced increases in [Ca2]i

levels in the DCD phase (Figures 1a and b). As NS8593 is a negative modulator for all KCa2 channels,10 we used the highly specic KCa2.2 channel blocker apamin. In line with ndings obtained with NS8593, apamin also promoted a further increase in [Ca2]i compared with the glutamate challenge alone (Figure 1c). Moreover, pre-incubation with apamin reduced the effect of NS309 on [Ca2]i

(Figure 1d).

Previous studies established NS309 as a potent activator of recombinant KCa2 channels that induces an increase of the

IAHP in hippocampal brain slices.12 Here we tested the effect of NS309 (50 mM) on IAHP in CA1 neurons from 17- to 21-day-old mice. Before NS309 application, the depolarization step induced a characteristic IAHP. NS309 induced an increase of

IAHP in all cells tested (six of six cells; Supplementary Figure S2). That was detectable even if the activator was removed from the bath (Supplementary Figure S2).

To further strengthen the direct action of KCa2 channels on calcium signaling, we investigated whether other potassium channels might affect calcium homeostasis. To this end, we applied diazoxide, a specic activator of KATP channels. Pre-treatment and also post-treatment with diazoxide did not affect glutamate-induced DCD, suggesting that KATP chan

nels do not have a direct effect on DCD signaling. In addition, inhibition of KATP channels with glibenclamide did not signicantly alter the DCD (Supplementary Figure S3).

As KCa2 channel activation reduced [Ca2]i after excessive stimulation of glutamate receptors in neurons, we next investigated whether KCa2 channel activation provided neuroprotective effects against glutamate-induced excitotoxi-city. Activation of KCa2 channels by NS309 signicantly reduced glutamate toxicity (Figure 1e). In contrast, when primary cortical neurons were subjected to toxic doses of glutamate in the presence of the KCa2.2 channel blocker apamin, neuronal cells were not rescued (Figure 1e). Further, apamin also attenuated NS309-induced neuronal survival indicating that neuroprotection by NS309 was mediated through KCa2.2 channel activation (Figure 1e). Although

NS8593 (50 mM) in combination with the glutamate challenge resulted in a further increase in DCD mean values compared with DCD induced by glutamate alone, NS8593 failed to show a further induction of apoptotic markers, such as caspase 3 activation (Supplementary Figure S3). As a positive control for a complete induction of cellular death we used ionomycin at different concentrations (Figure 1e). Notably, NS309 failed to promote cellular survival against glutamate toxicity when KCa2.2 channels were downregulated by siRNA, conrming the particular role of this subtype as previously demonstrated by pharmacological compounds (Supplementary Figure S4). Overall, these ndings strongly suggest that KCa2 channel activity was critical for the maintenance of intracellular Ca2

homeostasis and protection against glutamate-induced excitotoxicity in primary neurons.

Modulation of KCa2 channels during deregulated Ca2 homeostasis. To assess the effectiveness of KCa2 channel modulators on regulating glutamate-induced [Ca2]i

deregulation, we also applied NS309 and NS8593 after the onset of the glutamate challenge. When NS309 was applied to neurons after the onset of the early peak of [Ca2]i, a

signicant recovery of [Ca2]i was observed (Figures 2a and b). NS309 reduced [Ca2]i by B75% in the rst minute and by B90% after 5 min. In contrast, inhibition of KCa2

channels by NS8593 further elevated [Ca2]i by 30% in the rst 5 min and by 60% after 10 min (Figure 2b).

As demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, NS309 unequivocally protected the neurons against glutamate toxicity when applied before and up to 3 h after the onset of a glutamate challenge, in line with the restoration of calcium homeostasis (Figure 2c).

KCa2 channels reduce the Ca2 inux from the extracellular space. Although NS309 completely restored [Ca2]i when applied in the rst minutes after onset of glutamate exposure (Figures 2b and 3a), NS309 only partially attenuated the DCD when applied during the late phase of increased [Ca2]i (Figure 3b). The remaining question of why KCa2 channel activation failed to completely recover [Ca2]i applied after the onset of DCD prompted us to investigate the sources of [Ca2]i increases. To this end, ethylene glycol tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA; 4 mM) were applied to complex extracellular [Ca2]. Under these conditions, glutamate neither increased [Ca2]i nor induced DCD (Figure 3c), suggesting that extracellular Ca2 was

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Figure 2 Effect of KCa2 channel modulators after the onset of glutamate-induced delayed calcium deregulation. (a) Representative FURA-2 uorescence intensities from single cells stimulated with 20 mM glutamate (at 1 min). NS309 (50 mM) or NS8593 (50 mM) were applied at 4 min after onset of the glutamate challenge. (b) Kinetic proles of [Ca2]i values (S.E.M.) stimulated with glutamate (20 mM) and then treated with NS309 (50 mM), NS8593 (50 mM) at the indicated time (n 1520). (c) DAPI counting of

neurons challenged with glutamate (20 mM) in the presence or absence of NS309 (50 mM) applied as indicated (*Po0.05, **Po0.01 versus glutamate-treated neurons, ANOVA Scheffs test)

required for [Ca2]i deregulation. Complete removal of external Ca2 during the early glutamate-induced [Ca2]i peak led to a fast [Ca2]i recovery (Figures 3d and f). However, when extracellular Ca2 was depleted with EDTA after the onset of DCD, [Ca2]i recovery was delayed and

incomplete (Figure 3g). The low-sensitivity of DCD to extracellular Ca2 removal was similar to the previously observed failure of NS309 to completely restore the [Ca2]i

at late time points. The remaining [Ca2]i could be a result of delayed Ca2 release from intracellular stores from the

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Figure 3 Depletion of extracellular Ca2 prevents the formation of delayed calcium deregulation (DCD). Neuronal cells were loaded with FURA-2 and then stimulated with glutamate (20 mM). The KCa2 channel activator (50 mM NS309), or EGTA (4 mM) were applied to neurons 2 min (a) or 912 min (c) after exposure to glutamate. The inuences of NS309 (a, b, fi), EDTA (c, f and g) and EGTA (d and e) on the kinetic prole of [Ca2]i were evaluated prior and after the onset of DCD. Depicted are the kinetic proles of [Ca2]i (S.E.M.; n 1520). (h and i) The depletion of ER intracellular Ca2 pool was achieved with 100 mM thapsigargin prior to or after the onset of DCD

endoplasmic reticulum (ER). To deplete Ca2 from ER stores and to block sarco/ER Ca2-ATPase (SERCA) pumps, we applied thapsigargin and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ). High concentrations of thapsigargin (100 mM) mediated very small [Ca2]i

increases followed by complete [Ca2]i recovery within 10 min (Supplementary Figure S5A). Pre-treatment with thapsigargin did not inuence the glutamate-induced [Ca2]i deregulation (Figure 3h However, when KCa2 channels were activated after the onset of DCD, 100 mM thapsigargin completely blocked the protective effect of

NS309 on [Ca2]i (Figure 3i). Thapsigargin and BHQ at lower concentrations (10 and 25 mM, respectively) only partially blocked the KCa2 channel-mediated rescue of [Ca2]i (Supplementary Figure S5B). Altogether, these ndings suggest that the remaining delayed increases in [Ca2]i were attributed to extracellular and intracellular sources.

Activation of KCa2 channels attenuates NMDA-induced [Ca2]i deregulation. Glutamate-induced increases in [Ca2]i through the NMDA receptor has a dominant role

in [Ca2]i deregulation and in neuronal death.10,13 In primary neurons, NMDA increased [Ca2]i and shaped the Ca2 kinetics in a similar manner as glutamate (Supplementary Figure S6A). Pre-treatment with the NMDA antagonist MK801 attenuated the magnitude of the early glutamate-induced [Ca2]i peak and prevented the onset of DCD (Supplementary Figure S6B). In addition, MK801 partially restored [Ca2]i levels when added after the onset of

NMDA-induced DCD (Supplementary Figure S6C). These data supported the conclusion that the initial increase in [Ca2]i was mediated by NMDAR stimulation, whereas the following DCD was mediated by additional sources of Ca2. Activation of KCa2 channels by NS309 resulted in a complete recovery of [Ca2]i when applied before NMDA-induced [Ca2]i deregulation (Supplementary Figure S6A). However, NS309 only partially restored [Ca2]i when applied after the onset of NMDA-induced DCD (Supplementary

Figure S6D). Moreover, MTT analysis revealed that pre-treatment with NS309 rescued primary neurons from NMDA-induced neuronal death, suggesting that KCa2 channels affected NMDA receptor activity and attenuated NMDA-mediated excitotoxicity (Supplementary Figure S6E).

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Figure 4 Glutamate attenuates the KCa2.2 channel protein expression. (a) Western blot analysis of KCa2.2 channel expression at the indicated time points after glutamate exposure (20 mM). Representative immunoblots are shown at the upper part of the quantied data of KCa2.2 channels analyzed from different experiments (n 3, *Po0.05

versus non-treated PCN). (b) Neuronal cells were challenged with glutamate for 24 h with or without 30 min pretreatment with NS309 (50 mM). Representative immunoblots are shown at the upper part of the quantied data of KCa2.2 channels (n 3, *Po0.05 versus non-treated PCN). (c) MTT analysis of neurons treated with NS309 (50 mM)

0 h, 3 h, 5 h and 7 h after the application of glutamate (20 mM). *Po0.05, **Po0.01 versus glutamate-treated neurons were considered to be signicant, ANOVA Scheffs test. (d and e) mRNA analysis of KCa2 channel subtypes in the presence or absence of glutamate in (d) astrocyteneuron co-culture and in (e) pure cortical neuronal cultures

Glutamate neurotoxicity is associated with reduced KCa2

channel protein levels. To gain further insight into the relation between glutamate toxicity and KCa2 channel-mediated [Ca2]i regulation, we next investigated the expression levels of KCa2 channels after the glutamate challenge. Glutamate reduced the protein levels of KCa2.2

channels in a time-dependent manner (Figure 4a). These results suggested that the observed DCD after glutamate receptor stimulation and the failure of NS309 to rescue [Ca2]i levels when applied with delay was at least in part attributed to KCa2 channel degradation. In fact, the KCa2 channel activator NS309 prevented the glutamate-induced decrease in KCa2.2 channel expression (Figure 4b) and

NS309 protected neurons against glutamate toxicity when applied up to 3 h after the onset of glutamate challenge, which was perfectly in line with the observed protein

expression levels of KCa2.2 channels (Figure 4c). Further, EDTA application was able to block glutamate-induced KCa2.2 channel downregulation, suggesting that increased [Ca2]i mediated this effect (Supplementary Figure S7A). On the other hand, increase in [Ca2]i can activate Ca2-dependent genes, which could control the expression of KCa2.2 channels. Interestingly, short application of the selective glutamate receptor agonist NMDA induced an upregulation of KCa2.2 channel protein levels in the long term (Supplementary Figure S7B). This was potentiated in cells in which calpain activity was blocked, suggesting that Ca2-dependent activation of calpains negatively controls KCa2.2 channel protein levels.

Analysis of mRNA levels of KCa2 channel subtypes revealed that cortical neurons express all KCa2 subtypes: KCa2.1, KCa2.2 and KCa2.3 (Figures 4d and e). The KCa2.3

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Figure 5 KCa2 channel activator NS309 reduces infarct volume after transient focal cerebral ischemia. (a) Representative coronal brain slices obtained at 24 h after focal cerebral ischemia from vehicle control animals (left) and mice treated with 2 mg/kg NS309 right. The slices were stained with cresyl violet to identify unstained infarct areas as marked by the dashed line. (b) Quantication of total infarct volume determined 24 h after MCAo in mice treated with 0.2 or 2 mg/kg NS309 compared with vehicle-treated controls. (c) Neuroscore measured at indicated time points after middle cerebral artery occlusion (n 6, *Po0.05 versus non-treated mice. MannWhitney U-test)

channel is less abundant than KCa2.1 and KCa2.2 channel subtypes in both pure neuronal cultures (treated with cytosine arabinoside (CAF)) and in neuronastrocyte co-cultures (without CAF treatment). In the pure cortical neuronal cultures, KCa2.3 channels seemed to be expressed at lower levels compared with neuronastrocyte co-cultures, suggesting that KCa2.1 and KCa2.2 channels mediated the effects of NS309 on glutamate-induced calcium deregulation. Glutamate did not drastically affect the KCa2.1 and KCa2.2 mRNA expression (Figures 4d and e).

KCa2 channel activator NS309 reduces ischemic brain damage after experimental middle cerebral artery occlusion (MCAo) in mice. To translate our results from in vitro studies in which NS309 promoted neuroprotection against glutamate and NMDA toxicity, we next evaluated whether NS309 could provide neuroprotective effects in a model of ischemic brain damage in vivo. NS309 (0.2 and 2 mg/kg) was applied intraperitoneally 30 min before transient focal cerebral ischemia. Controls received vehicle, 2% DMSO in 0.9% saline. At 24 h after reperfusion, infarct volume was 697 mm2 (meanS.E.M.) in the control group (Figures 5a and b). Non-treated animals showed extensive ischemic injury, whereas signicant protection was observed in NS309-treated mice (Figure 5a). Histomorphometrical analysis demonstrated that a single dose of NS309 (2 mg/kg) reduced the infarct volume to 454 mm3 compared with the vehicle-treated controls (697 mm2;

meanS.E.M.; Figure 5b). The lower dose of NS309(0.2 mg/kg) did not reduce the infarct volume compared to the controls. NS309-treated mice (2 mg/kg) exhibited fast

and extensive recovery with improved neurological function score14 compared with controls as determined 24 h after MCAo (Figure 5c).

Discussion

In this study, we identied KCa2 channels as a promising target to preserve [Ca2]i homeostasis by counteracting glutamate- and NMDA-induced [Ca2]i deregulation and neurotoxicity in vitro and reducing infarct development after cerebral ischemia in vivo. In neurons, glutamate triggers immediate increases in [Ca2]i through activation of

NMDA-receptors followed by sustained disturbances in the intracellular Ca2 homeostasis.10,15 However, key regulators of intracellular Ca2 homeostasis that may serve as targets to prevent such sustained glutamate toxicity in neurological diseases have not been identied.

In this study, the KCa2 activator NS309 signicantly attenuated [Ca2]i increase when applied before the onset of DCD, and partially contributed to [Ca2]i recovery when applied after onset of glutamate-induced DCD. Further, we observed a close correlation of the neuroprotective potential of KCa2 channel activation and their expression levels.

Notably, activation of the KCa2 channels preserved their expression and their protective potential after exposure to glutamate or NMDA. Previous reports suggested that reduced expression and functional loss of KCa2 channels were connected to hyperexcitability in neurodegenerative processes.16,17 The exact mechanism of such KCa2 channel inactivation associated with neurodegenerative processes has not been claried so far. Our results now indicate that

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activation of NMDAR was involved in glutamate-induced [Ca2]i deregulation and subsequent delayed neuronal death. Both, NMDA inhibitors and pharmacological activation of KCa2 channels attenuated NMDA- and glutamate-induced [Ca2]i deregulation. In the soma of CA1 hippocampal regions, single-channel measurements showed that L-type calcium channel and KCa2 channels are co-localised.9 The contribution of voltage-dependent channels to the formation and maintenance of DCD is unlikely, as previous experiments using the L-type calcium channel blocker nitrendipine had little effect on the delayed Ca2 overload once DCD had been established.1 However, a more recent study showed the involvement of transient receptor potential and store-operated Ca2 channels, as the respective inhibitors 2-aminoethoxydiphenyl borate and La3 attenuated glutamate-induced DCD.18 In bullfrog sympathetic neurons, Ca2-induced Ca2 release activation prolongs Ca2 transients and activates BK channels that are closely associated with N-type Ca2

channels, ryanodine and KCa2 channels.9 Ryanodine and IP3 receptors might also be involved in DCD regulatory processes, as application of ryanodine or caffeine was shown to inhibit glutamate-induced DCD.18 Removing the extracellular Ca2 by adding the extracellular Ca2 chelators EDTA or EGTA resulted in maintenance of [Ca2]i. Thus, activation of KCa2 appears to be highly efcient to block pathological

Ca2 inux from the extracellular space in the context of glutamate excitotoxicity.

Pharmacological blockage of SERCA pumps with thapsigargin or BHQ partially blocked the protective effect of NS309 on DCD. However, thapsigargin alone did not alter [Ca2]i

and did not further exacerbate DCD, arguing against a major contribution of intracellular store depletion for the observed glutamate-induced DCD. Our observation is in agreement with a report describing that thapsigargin did not increase mean onset time or incidence of DCD.18 Overall, our ndings propose that NS309-mediated KCa2 activation restores Ca2 homeostasis by diminishing the Ca2 entry from the extra-cellular pool but still requires functional intracellular Ca2

stores for full effectiveness.

Another important nding was that NS309 preserved the expression of KCa2 channels in neurons exposed to glutamate. This suggests that glutamate-induced excitotoxi-city is mediated by activation of NMDAR and concomitant disruption of counteractive mechanisms such as KCa2

channels that disappear after the excitotoxic stimulus. This observation may explain both, the lack of adaptation to glutamate receptor overstimulation, and the therapeutic time window observed for NS309. As shown in this study, NS309 mediated neuroprotection only when applied up to 3 h after the onset of [Ca2]i deregulation. This is in agreement with the time window of the progressive decline in KCa2.2 channel expression levels upon glutamate damage. After exposure to glutamate for 5 h or more, neurons expressed less KCa2.2

channels and around this time point NS309 lost the neuroprotective potential. Apparently, such reduced KCa2.2

protein levels resulted in a failure to regulate neuronal excitability despite increased Ca2 concentrations that usually enhance the activity of the channel. Thus, neurons undergoing delayed neuronal death after the glutamate challenge were continuously deprived of this important feed-

back mechanism of [Ca2]i, therefore showing increased excitability and deregulated calcium homeostasis. These ndings are in agreement with the direct correlation between the prevention of DCD and reduced KCa2.2 channel degradation. The mechanism of KCa2 channel regulation is still unclear but may result from Ca2-dependent protein degradation or enhanced internalization from the membrane, which might be dependent on calpain activation.

Brief application of the glutamate receptor agonist NMDA, on the contrary, increased KCa2.2 channel protein levels, which was further augmented by calpain inhibition. This suggests that activation of calcium-dependent proteases and/ or Ca2-dependent gene transcription may regulate KCa2.2 channel expression physiologically. Although Ca2-activated calpains may enhance KCa2.2 degradation, nuclear factor-k B (NF-kB) activity may enhance neuroprotective KCa2.2

expression.19,20 It has been shown that preserved transcrip

tional activity of NF-kB in neurons provides cerebroprotective effects in a variety of degenerative conditions after acute brain damage.21,22 Thus, in addition to direct pharmacological

activation as achieved here with NS309, induction of KCa2

channel expression through enhanced NF-kB transcriptional activity may be an additional strategy to activate this system of intracellular Ca2 regulation.

The promising effects of KCa2 channel activation demonstrated in vitro are also relevant for ischemic neuronal death in vivo. Our data strongly suggest a therapeutic potential for KCa2 channel activators in paradigms of excitotoxic neuronal damage that contributes to infarct development after cerebral ischemia. In line with our ndings, overexpression of KCa2

channels in the dentate gyrus attenuated kainic acid-induced hippocampal CA3 lesion, suggesting that KCa2 channels are a common motif of Ca2 autoregulation at different glutamate receptors.23 In vivo, the protective function of NS309 may be extended to non-neuronal cells, as NS309 can enhance ATP-evoked membrane hyperpolarization, along with acute endothelial NO synthesis in isolated endothelial cells. In addition, NS309 was shown to augment the acetylcholine-induced vasodilatation in small-resistance arterioles that might improve the recovery of damaged tissue.24 It is

interesting to note that two FDA-approved drugs, namely, riluzole (used in the treatment of amyotrophic lateral sclerosis) and chlorzoxazone (used as central myorelaxant)25 have

been reported to enhance the activity of KCa2 channels,26 suggesting a clinical relevance of the present ndings on neuroprotection mediated by KCa2 channel activation in vitro and in vivo. For example, KCa2 channel activation may signicantly contribute to the reported effects of the neuroprotectant riluzole, that is, that it inhibits the release of glutamate from nerve terminals, modulates NMDA receptors and reduces neuronal excitability.27

In conclusion, our data suggest that activation of KCa2

channels promotes neuroprotection in vitro and in vivo by reducing glutamate- and NMDA-induced [Ca2]i deregulation. KCa2 channels may, therefore, have a major role in the regulatory feedback loop that interrupts the glutamate-triggered neuronal hyperexcitability, progressive disturbance of Ca2 homeostasis and excitotoxic neuronal death. Accordingly, KCa2 channels may serve as novel therapeutic targets to prevent intracellular Ca2 overload under

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pathological conditions associated with glutamate-induced excitotoxicity.

Materials and MethodsPrimary cortical neuron culture. Primary cortical neurons were plated at a density of 16 103 cells/well (96 well plates) and 3 105 cells/well (6 well plates)

on polyethyleneimine pre-coated plates. Neurobasal medium supplemented with 5 mM HEPES, 1.2 mM glutamine, 2% (v/v) B27 supplement (20 ml/l) and gentamicin(0.1 mg/ml) was used as a culture medium. Neurons were treated with the KCa2

channel activator NS309, the KCa2 channel blocker NS8593 or apamin for the indicated time periods. On the basis of previous kinetic studies, NS309 was used at a concentration of 50 mM.19

Evaluation of cell viability. Neuronal viability was determined by the colorimetric MTT assay. The absorbance of each well was determined with an automated FLUOstar Optima reader (BMG Labtech, Offenburg, Germany) at 570 nm with a reference lter at 630 nm.

Calcium measurements in single neurons using calcium imaging. Primary cortical cells were incubated with 2 mM FURA-2 AM for 30 min at 371C in HEPES-ringer buffer. Drugs were diluted in HEPES-ringer buffer (20 mM glutamate, 500 mM NMDA, 50 mM NS309, 50 mM NS8593, 1 mM apamin, 25 mM MK801, 4 mM EDTA and 4 mM EGTA). Fluorescence intensities from single cells excited at the two wavelengths (F340 and F380) were recorded separately and combined (uorescence ratio: r F340/F380) after background subtraction

(uorescence of a cell-free area).

Protein analysis. Primary cortical neurons were lysed in 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, complete mini protease inhibitor cocktail tablet and phosphatase inhibitor cocktail 1 and 2. The membranes were incubated overnight with primary antibodies (1 : 3000; rabbit anti-KCa2.228 channel at 41C and afterwards with peroxidase-conjugated secondary antibodies (1 : 2500).

RT-PCR. Total RNA was extracted using the NucleoSpin RNA II kit (Macherey-Nagel GmbH & Co. KG, Dren, Germany). RT reactions were conducted in a thermo cycler with setting at 421C for 30 min. Amplications using specic primers29

by PCR were carried out for 27 or 30 cycles at various steps: (1) denaturing at 951C for 4 min; (2) 941C for 30 s; (3) annealing temperature (Tm) for 30 s, depending on the KCa2 isoform of interest; and (4) extension at 721C for 30 s. The nal extension step was set to 721C for 5 min. The Tm for KCa2.1 was 631C, 57,31C for KCa2.2 and 611C for KCa2.3.

Transient focal cerebral ischemia. All animal experiments were conducted according to the guidelines of Government of Upper Bavaria. Male C57BL/6 mice were subjected to 60 min transient MCAo by an intraluminal lament as previously described.14 Mice were killed and brains were removed and frozen in powdered dry ice 24 h after reperfusion. Infarct volume was calculated by multiplying the infarct areas with the distance between sections. NS309 was administered 30 min before MCAo by intraperitoneal injection (100 ml per 20 g) at a concentration of 0.2 mg/kg and 2 mg/kg. Saline (0.9%) with 2% DMSO was used as vehicle.

Statistical analysis. All data are given as meansS.D. For statistical comparisons between two groups, Students t-test was used; multiple comparisons were performed by ANOVA followed by Scheffs post hoc test. Calculations were performed with the Winstat standard statistical software package. For the MCAo experiments we have used the MannWhitney U-test for the analysis of differences between groups and also by ANOVA followed by Scheffs or Tukeys as post hoc analysis. A statistically signicant difference was assumed at Po0.05.

Other methods, including hippocampal slice preparation, patch-clamp recordings and data sanalysis were carried out as described in Andres et al.29, Li et al.30, Pedarzani et al.31 and Landshamer et al.32 Further details are provided in Supplementary Materials and Methods.

Conict of interest

The authors declare no conict of interest.

Acknowledgements. We thank Hermann Kalwa and Alexander Dietrich for their assistance in calcium imaging and Sandra Engel and Uta Mamrak for excellent technical assistance, and Emma Jane Esser for careful reading and corrections of the manuscript. UE and IN were supported by the European Unions FP6 funding, NeuroproMiSe, LSHM-CT-2005-018637. This work reects only the views of the author. The European Community is not liable for any use that may be made of the information herein.

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