Supporting information

Materials and Methods

Primary cortical neuron culture

Primary cortical neurons were prepared from embryonic brains (E16-18) of rats. Meninges were removed and the cortical neurons separated by mechanical dissociation and mild trypsinisation. Cells were plated at a density of 16 x 103 cells/well (96 well plates) and 3 x 105 cells/well (6 well plates) on polyethyleneimine pre-coated plates. Neurobasal medium (Invitrogen, Karlsruhe, Germany) supplemented with 5mM HEPES, 1.2mM glutamine, 2% (v/v) B27 supplement (Invitrogen, Karlsruhe, Germany, 20ml/l) and gentamicin (0.1mg/ml) was used as a culture medium. After 48 h, neurons were treated with 1 µM cytosine arabinoside (CAF) for another 48 h to inhibit non-neuronal cell growth. After 6-7 days of in vitro culture, the neurons were used for experiments. Application of siRNA for KCa2.2 channels (250nM) was performed on day 4 till day 6 using Accell SMART pool for KCNN2 (Cat no E-049792-00, Thermo Scientific, Dharmacon, Germany).

Neurons were treated with the KCa2 channel activator NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime), the KCa2 channel blocker NS8593 (N-[(1R)-1,2,3,4-tetrahydro-1-naphthalenyl]-1H-benzimidazol-2-amine hydrochloride) or apamin for the indicated time periods. Based on previous kinetic studies, NS309 was used at a concentration of 50 µM (19).

For a better understanding of KCa2 channel contribution to neuronal functions, we used the KCa2 channel modulators NS309 and NS8593 (9). The compound NS309 represents the most potent positive gating modulator of the KCa2 channels available. NS309 stabilizes the interaction between calmodulin (CaM) and KCa2 channels, thereby enhancing the coupling between Ca2+ binding and the gating of KCa2 channels (30). NS309 has been shown to be a potent activator of recombinant KCa2 channels and induces an increase of the afterhyperpolarization current in hippocampal brain slices (31). Since these modulators are not selective for any of the KCa2 channel subtypes, we also applied apamin, a bee venom-derived KCa2 channel inhibitor that exerts the highest binding affinity to the KCa2.2 channel subtype as compared to lower binding affinities to KCa2.3 and KCa2.1 channel subtypes (9). These negative modulators have been reported to decrease the Ca2+ sensitivity of KCa2 channels (11).

NS309 and NS8593 were prepared as stock solution in DMSO. The final concentration of DMSO in all experiments was less than 0.5% vol/vol. At this concentration, DMSO had no effect on glutamate-induced cell death. All pharmacological substances were purchased from Sigma (Sigma-Aldrich, Deisenhofen, Germany).

To induce glutamate excitotoxicity, neurons were exposed for 24 h (or otherwise indicated) to 20 µM glutamate. Experiments were repeated at least three times and analyses were performed without knowledge of the treatment history.

Evaluation of cell viability

Twenty-four hours after glutamate treatment, neuronal viability was determined by the colorimetric MTT assay as described previously (32). Briefly, 0.5 mg/ml MTT solution was incubated for 1h. Cells were frozen for at least 2 h at -80°C followed by elution with DMSO solution. The absorbance of each well was determined with an automated FLUOstar Optima reader (BMG Labtech, Offenburg, Germany) at 570 nm with a reference filter at 630 nm. In addition to the MTT analysis, we performed DAPI staining of the nuclei 18 h after onset of treatment. Images were obtained using a fluorescence microscope (DMI6000, Leica, Germany). Pycnotic nuclei representing apoptotic cells were counted at 10 different spots of each 35mm dish (~2000 cells per condition, n=5).

Calcium measurements in single neurons using calcium imaging

Primary cortical cells were incubated with 2 µM FURA-2 AM for 30 min at 37°C in HEPES-Ringer buffer (HRB; 136.4 mM NaCl/5.6 mM KCl/1 mM MgCl2/2.2 mM CaCl2/10 mM HEPES/5 mM glucose/0.1% BSA, pH 7.4). Drugs were diluted in HEPES-Ringer buffer (20 µM Glutamate, 500 µM NMDA, 50 µM NS309, 50 µM NS8593, 1 µM apamin, 25 µM MK801, 4 mM EDTA and 4 mM EGTA). Images were acquired using a Polychrome II monochromator and an IMAGO CCD camera (Till Photonics, Martinsried, Germany) coupled to an inverted microscope (IX70; Olympus, Hamburg, Germany). Images were collected with 20x0,8 numerical apeture (NA) oil immersion objective. Fluorescence intensities from single cells excited at the two wavelengths (F340 and F380) and the emission used was 510nm. An increase in intracellular Ca2+ was reflected by a fluorescence increase when exciting at 340 nm and a corresponding decrease with excitation at 380 nm. F340 and F380 were recorded separately and combined (fluorescence ratio: r = F340/F380) after background subtraction (fluorescence of a cell-free area).

To study the long-term effects of glutamate on [Ca2+]i, we used a FluoStar Optima system (BMG Lab Technology, GmbH, Offenburg, Germany). Neurons were loaded for 30min with 2µM Fluo-4 AM (Molecular Probes, Eugene, OR) in a solution containing 0,005% pluronic acid and 2µM probenecid (Invitrogen, Karlsruhe, Germany). The excitation/emission filter pair 485/520 nm was used in the experiments.

RT-PCR

Total RNA was extracted using the NucleoSpin RNA II kit (Macherey-Nagel GmbH & Co. KG; Dürel, Germany). Reverse transcription (RT) reactions were performed using SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase kit (Invitrogen, Karlsruhe, Germany).

RT reactions were conducted in a Thermo Cycler (SensoQuest GmbH, Gottingen, Germany) with setting at 42° C for 30 min. Amplifications using specific primers (29) by PCR were carried out for 27 or 30 cycles at various steps: (1) denaturing at 95° C for 4 min; (2) 94° C for 30 sec; (3) Tm (annealing temperature) for 30 sec, depending on the KCa2 isoform of interest;(4) extension at 72° C for 30 sec. The final extension step was set to 72° C for 5 min. The Tm for KCa2.1 was 63° C, 57,3° C for KCa2.2, 61° C for KCa2.3.

Protein analysis
Primary cortical neurons were 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 (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich, Deisenhofen, Germany). The samples were centrifuged at 9,000 g for 10 min. Twenty micrograms of total protein were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a PVDF membrane (Millipore Corporation, Billerica, MA, USA). The membranes were incubated overnight with primary antibodies (1: 3,000; rabbit anti-KCa2.2 channel (28), caspase 3 (Cell Signaling) at 4ºC and afterwards with peroxidase-conjugated secondary antibodies (1: 2,500). Proteins were detected with an ECL detection system according to the manufacturer’s instructions (Applied Biosystems, Bedford, MA, USA). Tubulin (1:200,000 antibody dilution) was used as an internal control to correct for variations in protein content.

Transient focal cerebral ischemia

All animal experiments were conducted according to the guidelines of Government of Upper Bavaria. Male C57BL/6 mice (19-22g; Charles River Laboratories, Sulzfeld, Germany) were subjected to transient MCAo by an intraluminal filament as previously described (14). Briefly, mice were anesthetized with 1.2% isoflurane in 33% oxygen and 65.8% N2O administered via a face mask. For MCAo, the left common carotid artery was exposed and a silicone-coated monofilament was introduced into the internal carotid artery and advanced distally until rCBF in the MCA territory measured by Laser-Doppler-Fluxmetry (Perimed, Sweden) decreased to less than 20% of baseline. Animals were then allowed to wake up in an incubator heated to 33°C. Animals were re-anesthetized and the filament was removed after 45 min MCAo. Mice were sacrificed and brains were removed and frozen in powdered dry ice 24 h after reperfusion. Ten micrometer thick coronal sections were collected every 750 µm throughout the brain. The resulting 11 sections were stained with cresyl violet and the area of infarct quantified in each section by digital image analysis. Infarct volume was calculated by multiplying the infarct areas with the distance between sections. An investigator without the knowledge of the treatment groups performed all measurements and calculations of infarct volume.

NS309 was administered 30 min before MCAo by intraperitoneal injection (100µl/ 20g) at a concentration of 0.2 mg/kg and 2 mg/kg. Saline (0.9%) with 2% DMSO was used as vehicle.

Hippocampal slice preparation

Acute brain slices were prepared from young male C57BL/6 mice of postnatal ages between 17 and 21 days. Mice were anesthetized with isoflurane (Baxter, Unterschleißheim, Germany) and then decapitated. The brain was quickly removed from the skull, immersed in ice-cold, cutting solution containing (in mM): 87 NaCl, 25 NaHCO3, 25 glucose, 75 sucrose, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2 and 7 MgCl2, equilibrated with 95% O2 and 5% CO2.

Thin horizontal slices (200 µm) of the hippocampal area were prepared. After cutting, slices were maintained submerged in a holding chamber filled with gassed artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 25 NaHCO3, 10 glucose, 2.5 KCl, 1.4 NaH2PO4, 2 CaCl2, 4 MgCl2 and 1.3 ascorbic acid and allowed to recover for 30 min at 35° C before being used for experiments. Slices were kept at room temperature for up to 6 h.

After the recovery period, single slices were transferred to a submerged recording chamber and superfused continuously (2.5 – 3.5 ml/min at room temperature) with ACSF containing (in mM): 125 NaCl, 25 NaHCO3, 1.25 KCl, 1.25 KH2PO4, 2.5 CaCl2, 1.5 MgCl2, and 16 glucose, equilibrated with 95% O2 and 5% CO2 (34).

Patch-clamp recordings and data analysis

Patch pipettes were pulled from borosilicate glass (Science Products, Hofheim, Germany) and filled with an internal solution containing (in mM): 135 potassium gluconate, 10 KCl, 10 HEPES, 2 Na2ATP, 1 MgCl2, and 0.4 Na3GTP, pH 7.2 with KOH. Patch pipettes had resistances of 3 - 5.5 MΩ when filled with the intracellular solution.

Whole-cell patch-clamp recordings were obtained from CA1 hippocampal neurons visualized by infrared differential interference contrast (IR-DIC) video microscopy with a IR CCD camera (VX55, TILL Photonics GmbH, Gräfelfing, Germany) mounted on an upright microscope (BX51WI, Olympus, Hamburg, Germany). Recordings were made using an EPC-10 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany).

Experiments with series resistances < 20 MW were accepted in the study and 50% of the series resistance was compensated electronically. Data were acquired using PULSE (HEKA Elektronik, Lambrecht, Germany). In voltage-clamp experiments, cells were held at a potential of -50 mV. Tail currents were evoked by 100 ms-depolarizing pulses to +45 mV every 30 s. Pulse data were acquired with a sampling rate of 2.0 kHz. In whole-cell voltage-clamp recordings, we measured responses to 100 ms – depolarizing pulses from -50 mV to +45 mV.

In all experiments, tetrodotoxin (TTX; 500 nM, Alomone Labs, Jerusalem, Israel) and tetraethylammonium chloride (TEA-Cl; 1 mM, Sigma-Aldrich, Deisenhofen, Germany) were added to the superfusing ACSF. NS309 (50 µM) was applied to the slice via the bath solution with the superfusate. Analysis and plotting were carried out using IgorPro 6.03 (WaveMetrics).

Supplemental figures

Figure S1

Figure 1. KCa2 channel activator NS309 reduces glutamate-mediated DCD. A. Primary cortical neurons were loaded with Fluo-4 AM in the presence of pluronic acid and probenecid and continuously monitored for 1 h with the FluoStar Optima system. NS309 was applied to neurons 30 min prior to [Ca2+]i measurements. Neurons were stimulated with glutamate (20 µM). Depicted are the kinetic profiles of [Ca2+]i values (±SEM) stimulated with glutamate with or without NS309 (50 µM). B. Neurons pre-treated with TTX (10 µM) did not reduce the glutamate-induced DCD.


Figure S2

Figure 2. NS309 is a positive modulator of the KCa2 channel-mediated neuronal IAHP. This figure shows the activating effect of NS309 on tail currents measured in a mouse CA1 neuron activated by 100 ms-depolarizing pulses from a holding potential of -50 mV to 45 mV. In the upper panel the tail current is shown before application of NS309 (control, black). The middle panel illustrates the increase of IAHP one minute (light grey) and 4 min (dark grey) after application of NS309 (50 µM) for 70 s (duration of NS309 application). The lower panel shows an overlay of the tail currents before (control, black) and after NS309 application (dark grey).


Figure S3

Figure 3. Pharmacological modulation of KATP channels does not directly influence glutamate-induced DCD. Neuronal cells were loaded with FURA-2 AM and then stimulated with glutamate (20 µM). (A) Neurons pre-treated or (B) post-treated with diazoxide (750 µM) showed similar deregulation in Ca2+ homeostasis as glutamate challenge (20 µM). C. Blockage of KATP channels with glibenclamide (10 µM) did not affect the glutamate-induced DCD. D. Cortical neurons were treated with NS8593 (50 and 75 µM) for 24h and caspase 3 was detected by western blot analysis.


Figure S4

Neuroprotective effect of NS309 is dependent on KCa2.2 channel expression. A. Cortical neurons were treated for 48h with specific siRNA for KCa2.2 channels (250nM). Afterwards, the neuroprotective effect of NS309 was tested in a glutamate toxicity model. Results shown represent mean ± SEM. (*p<0.05 versus glutamate-treated neurons, ANOVA, Scheffé test).
Figure S5

Figure 5. Depletion of intracellular Ca2+ pool does not alter the formation of delayed calcium deregulation (DCD). A. Neurons treated with thapsigargin (100 µM) showed a slight increase in [Ca2+]i followed by a fast [Ca2+]i recovery. B. DCD was induced by glutamate (20 µM). KCa2 channel activator NS309 (50 µM) was applied to neurons after the onset of DCD, BHQ (25 µM) was applied during the NS309-induced [Ca2+]i recovery phase and showed slight improvement of DCD values.


Figure S6

Figure 6. Effect of KCa2 channel activators on NMDA-induced delayed calcium deregulation. Neuronal cells were stimulated with (A, C, D) NMDA (500 µM) or (B) glutamate in the presence of (A, D) NS309 (50 µM) or (B, C) MK801 (25 µM) (A, B) before the onset of DCD or (C, D) after the onset of DCD (n=15-20). E. Analysis of viability of neurons challenged with NMDA in the presence or absence of NS309. Each bar represents the mean of 6 absorbance values of a single experiment. Experiments were repeated at least 3 times. Results shown represent mean ± SEM. (*p<0.05 versus NMDA-treated neurons, ANOVA, Scheffé test).