The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA). The specificity of the anti-rEag1 and the anti-rEag2 antibodies has been previously verified .
Animals and hippocampal cultures
All procedures were in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council 2003) and approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University.
15-day pregnant Sprague–Dawley rats were purchased from the Facility for Animal Research of the National Yang-Ming University. Dissociated hippocampal cultures were prepared using a previously described protocol  with a minor modification . In brief, hippocampi were dissected from the brains of embryonic day 18 (E18) embryos, the brains of which were removed and placed in the Hank’s balanced salt solution that contains 10 mM HEPES (pH 7.4) and 1 mM sodium pyruvate. The hippocampus was dissected out and dissociated by incubation with 0.25% trypsin solution. The dissociated cells were plated on coverslips at a density of 200 and 1000 cells/mm2 for immunofluorescence and DNA transfection, respectively. Coverslips were coated with poly-D-lysine (1 mg/ml) (Sigma) and laminin (15 μg/ml) (Sigma). Cultures were maintained in the Neurobasal media supplemented with B27 (2%) and glutamax I (0.5 mM) (Invitrogen) in a humidified 5% CO2 incubator at 37°C.
Adult female Xenopus laevis (African Xenopus Facility) were anesthetized by immersion in ice water containing Tricaine (1.5 g/liter). Ovarian follicles were removed from Xenopus frogs, cut into small pieces, and incubated in the ND96 solution [(in mM) 96 NaCl, 2 KCl, 1.8 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.2]. To remove the follicular membrane, Xenopus oocytes were incubated in the Ca2+-free ND96 solution containing collagenase (2 mg/ml) on an orbital shaker (~200 rpm) for about 60-90 min at room temperature. After several washes with collagenase-free, Ca2+-free ND96, oocytes were transferred to ND96. Stage V-VI Xenopus oocytes were then selected for cRNA injection.
The cDNAs for rEag1 and rEag2 K+ channel subunits were kindly provided by Dr. Olaf Pongs (Institute fur Neurale, Signalverarbeitung, Zentrum fur Molekulare Neurobiologie). Green fluorescent protein (GFP)-tagged rEag1 and rEag2 constructs were made by subcloning the full length rEag1 and rEag2 cDNAs into the pEGFP mammalian expression vector (Clontech).
The design of the chimeras between rEag1 and rEag2 were based on sequence alignment. Chimeric channels were constructed by using the overlap PCR mutagenesis method. All constructs were verified by DNA sequencing (Genome Research Center, National Yang-Ming University).
For DNA transfection, human embryonic kidney (HEK) 293 T cells were maintained in DMEM (Invitrogen) supplemented with 2 mM L-glutamine, 100 units/ml penicillin/streptomycin, and 10% (v/v) fetal bovine serum (Hyclone). For immunofluorescence and electrophysiology, cells were grown on poly-lysine-coated coverslips. After 24 hrs, HEK293T cells were transiently transfected with cDNAs by using the Lipofectamine 2000 (LF2000) reagent (Life Technologies).
Cultured hippocampal neurons at 7 days in vitro (DIV7) were also transfected by using LF2000. Briefly, various expression constructs were incubated with the LF2000 reagent for 20 min at room temperature. DNA-lipofectamine diluted in the complete medium was added to neuron culture wells. After 4-hr incubation at 37°C under 5% CO2, cells were washed gently three times with the culture media and maintained in the incubator before being examined under a fluorescence microscope.
For in vitro transcription, cDNAs were linearized with NotI. Capped cRNAs were transcribed in vitro from the linearized cDNA template with the mMessage mMachine T7 kit (Ambion). The apparent molecular weight and concentration of cRNAs were verified with gel electrophoresis and determined by spectrophotometry, respectively. For cRNA injection, the total volume of injection was always 41.4 nl per Xenopus oocyte. Injected oocytes were stored at 16°C in ND96.
Coverslips containing HEK293T cells or hippocampal neurons were rinsed in PBS [(in mM) 136 NaCl, 2.5 KCl, 1.5 KH2PO4, 6.5 Na2HPO4, pH 7.4] and then fixed with 4% paraformaldehyde in PBS at 4°C for 20 min. Cells were then permeabilized and blocked with a blocking buffer (5% normal goat serum in 20 mM phosphate buffer, pH 7.4, 0.1% (v/v) Triton X-100, and 0.45 M NaCl) for 60 min at 4°C. Appropriate dilutions of primary antibodies were applied in the blocking buffer overnight at 4°C. Immunoreactivities were visualized with goat-anti-mouse antibodies conjugated to Alexa568 or with goat anti-rabbit antibodies conjugated to Alexa488 (Molecular Probes). The fluorescence images were viewed and acquired with a Leica TCS SP5 laser-scanning confocal microscope.
Image analyses were performed with the ImageJ software (National Institute of Health). To determine the number of immunofluorescence clusters per fixed length of neurite, built-in “set scale” and “freehand tool” functions of the software were applied to trace multiple 100-μm neurite segments, followed by counting the number of PSD-95/rEag1/rEag2 puncta within each 100-μm neurite segment. Co-localization of PSD-95 (appearing as red punctate pixels) and rEag1/rEag2 (appearing as green punctate pixels) puncta within each 100-μm neurite segment was recognized by identifying the presence of overlapping punctate pixels. For neurons transfected with various GFP-tagged constructs, the number of GFP puncta per neuron was also estimated using ImageJ. Statistical analyses were executed with the Origin 7.0 software (Microcal Software). All numerical data are shown as mean ± standard error (SEM).
Subcellular fractionation of rat brain and preparation of PSDs
Subcellular and PSD fractions of adult rat brains were prepared as described previously . In brief, adult rat forebrains were homogenized in the buffer H1 [(in mM) 320 sucrose, 1 NaHCO3, 0.5 CaCl2, 0.1 PMSF] containing a cocktail of protease inhibitors (Roche) and centrifuged at 1,400×g to remove nuclei and other large debris (P1). The S1 fraction was subject to centrifugation at 13,800xg to obtain the crude synaptosome fraction (P2). The pellet was resuspended in the buffer H2 [(in mM) 0.32 M sucrose and 1 mM NaHCO3)] and layered onto the top of the discontinuous sucrose density gradient by using 0.85, 1.0, and 1.2 M sucrose layers. The gradient was centrifuged at 65,000xg for 2 hrs in a Beckman Instruments SW-28 rotor and the synaptosomal fraction (SPM) was recovered from the 1.0-1.2 M sucrose interface. The synaptosomal fraction was extracted in ice-cold 0.5% Triton X-100/50 mM Tris–HCl (pH 7.9) for 15 min and centrifuged at 32,000xg for 45 min to obtain the PSD I pellet. The pellet was resuspended and further extracted a second time with 0.5% Triton X-100/50 mM Tris–HCl (pH 7.9), followed by centrifugation at 200,000×g for 45 min to obtain the PSD II pellet. Protein concentration was determined by the BCA protein assay kit (Thermo). For immunoblotting, 25 μg (H, S1, P2, and SPM) or 5 μg (SPM, PSD I, and PSD II) of proteins were separated by SDS-PAGE, blotted onto nitrocellulose membranes, incubated with the primary antibodies, and imaged with the enhanced chemiluminescence method (Thermo).
For HEK293T cells, conventional whole-cell patch clamp technique was used to record Eag K+ currents as described previously . In brief, recordings were performed at 24-48 hrs post-transfection. Patch electrodes with a resistance of ~4 MΩ were pulled on a Narishige PP-830 electrode puller and were filled with a solution containing (in mM) 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, pH 7.2. External bath solution comprised (in mM) 140 NaCl, 5 KCl, 1 CaCl2, and 10 HEPES, pH 7.2.
Conventional two-electrode voltage clamp recording in Xenopus oocytes were performed as described previously . In brief, 2-3 days after cRNA injection, oocytes were functionally assayed in a recording bath containing about ~200 μl of the Ringer solution [(in mM): 115 NaCl, 3 KCl, 1.8 CaCl2, 10 HEPES, pH 7.2]. An agarose bridge was used to connect the bath solution with a ground chamber (containing 3 M KCl) into which two ground electrodes were inserted. Borosilicate electrodes (0.1 –1 MΩ) used in voltage recording and current injection were filled with 3 M KCl.
Voltage-clamp protocols were applied with the pCLAMP 8.2/9.0 software (Molecular Devices). Data were acquired with an Axopatch 200A amplifier (Molecular Devices) (for HEK293T cells) or OC-725C oocyte clamp (Warner) (for Xenopus oocytes), followed by digitization at 10 kHz with the Digidata 1320A/1322A system (Molecular Devices). Also by using the pCLAMP 8.2/9.0 software, data were filtered at 1 kHz and passive membrane properties were compensated with the -P/4 leak subtraction method. All recordings were performed at room temperature (20-22°C).
Cells with large currents in which voltage clamp errors might appear were excluded from data analyses. Kinetic fitting of Eag K+ current traces were implemented with the pCLAMP 8.2/9.0 software. Subsequent numerical analyses and data plotting were performed with the Origin 7.0 software. All numerical data are shown as mean ± SEM.