Chemicals
Spadin was purchased from Tocris (Tocris Bioscience, Bristol, UK). Barium, Triethylamine (TEA) and Apamin were purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA). Kainic acid (KA) was purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA). All substances were stored as stock solutions at − 20°C and diluted to the required concentrations in standard bath solution immediately prior to experimentation.
Animals
Transgenic NHERF-1 mouse was purchased in Jackson Laboratory (stock No: 012862). Littermates including NHERF-1 wild-type (WT) or heterozygote knockout (Het) mice were used for this study. Mice were housed and maintained in standard laboratory conditions of 12:12 h light: dark cycle. Regular chow and water were provided ad libitum. Male C57BL/6N mice 7–12 weeks-old were used for all experiments. Animal care and handling were performed according to the institutional guidelines of Institutional Animal Care and Use Committee (IACUC) at Korea University (Seoul, Korea) and at the Korea Institute of Science and Technology (Seoul, Korea). The animals were genotyped by PCR of tail DNA samples. The following primers were used: NHERF-1 common forward primer − 5′-GAGAAGGGTCCAAATGGCTA-3′; NHERF-1 WT reverse primer − 5′-TTCGGCCTCATTCTGGTC-3′ and NHERF-1 KO reverse primer − 5′-CGCCTTCTTGACGAGTTCTT-3′
HEK293T cells culture and transfection
HEK293T cells were purchased from the Korean Cell Line Bank (Seoul National University, Seoul, Korea) and cultured in DMEM (Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37℃ in a 5% CO2 incubator. Transfection of expression vectors was perfomed with Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA), according to the manufacturer’s protocol.
Primary astrocyte culture and transfection
Primary astrocytes were maintained in DMEM supplemented with 10% fetal bovine serum, 10% horse serum, and penicillin–streptomycin. Cultures were maintained at 37°C in a 5% CO2 incubator. After 4 days, cells were washed by repeated pipetting, and the media was replaced to remove debris and other cells. For gene silencing or overexpressing, cultured astrocytes were transfected with Nherf-1 shRNAs or GFP-NHERF-1 with an optimized voltage protocol (1,300 V, 20 pulse width ms, 2 pulses number) using the Neon Electroporation instrument (Thermo Fisher, Waltham, MA, USA).
Construction of expression vectors
cDNAs encoding for full-length mouse TREK-1 (GenBank accession no. NM_010607), mouse TWIK-1 (NM_008430) and mouse NHERF-1 (NM_012030) were obtained by using an RT-PCR-based gateway cloning method (Thermo Fisher, Waltham, MA, USA). Deletion mutants of TREK-1 and NHERF-1 were also generated using full-length cDNAs as templates via EZchange site-directed mutagenesis kit (Enzynomics, Daejeon, Korea). All constructs were cloned into various vectors by gateway cloning.
Construction of shRNAs
For gene silencing in cultured astrocyte, Trek-1 shRNA were constructed and validated as described previously 7. The target region of Nherf-1 shRNA is 5’-AGCGATACCAGTGAGGAGCTAAAT-3’
Construction of viral vectors
For Nherf-1 gene or TREK-1 N4 overexpression in vivo, the mouse NHERF-1 or 2A-mCherry, TREK-1 N4 or 2A-GFP was re-cloned into pDONR207 P1P5R or pDONR207 P5P2 vectors respectively via two independent BP-reactions (ThermoFisher, Waltham, MA, USA). These vectors were cloned into the pAAV-GFAP promoter vector using Multisite Gateway LR recombination reaction (ThermoFisher, Waltham, MA, USA). AAV viral vectors were produced by KIST virus facility (Korea).
Co-immunoprecipitation (Co-IP) and immunoblotting
For co-immunoprecipitation in overexpressed HEK293T cells or endogenous astrocytes, whole-cell lysates were mixed overnight at 4 ℃ with 1 mg/ml anti-HA (Santa Cruz Biotechnology, Dallas, TX, USA, cat# sc-7392), anti-GFP (Santa Cruz Biotechnology, Dallas, TX, USA cat# sc-9996) or anti-NHERF-1 (H-100; Santa Cruz Biotechnology) antibodies in lysis buffer (50mM Tris–HCl (pH 7.4), 150mM NaCl, 2mM EDTA, 1mM PMSF, 1% Triton X-100 and 1% NP-40) containing a protease-inhibitor cocktail (Roche). Immune complexes were incubated by binding to mixed protein A/G PLUS Agarose (Santa Cruz Biotechnology) for 1 h and then washed four times with lysis buffer. For immunoblotting, protein samples were separated by SDS–PAGE using 10% gels. The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-rad). The blots were incubated overnight at 4 ℃ with anti-HA antibody (Roche Applied Science, Penzberg, Germany cat# 11867431001, 1:1,000), anti-GFP (Santa Cruz Biotechnology, Dallas, TX, USA cat# sc-9996, 1:1,000), anti-TREK-1 (Alomone Labs, Jerusalem, Israel, cat#APC-047, 1:500) or anti-NHERF-1 (Thermo Fisher, Waltham, MA, USA, cat# PA1-090, 1:1,000). Blots were then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse (Jackson ImmunoResearch, West Grove, PA, USA, 1:3000), anti-rat (Jackson ImmunoResearch, West Grove, PA, USA, 1:3000), or anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA, 1:3000) followed by washing and detection of immunoreactivity with enhanced chemiluminescence (Bio-rad, Hercules, CA, USA).
Cell surface biotinylation
Biotinylation assays were performed using Sulfo-NHS-SS-Biotin (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. Nherf-1 shRNA, Scramble shRNA, GFP control or GFP-NHERF-1-transfected astrocytes were incubated at 4℃ and washed three times with PBS. Then, cells were incubated with Sulfo-NHS-SS-Biotin in PBS for 30 min at 4℃. After washing cells with ice-cold PBS containing glycine, non-reacted biotinylation reagent was removed by lysing cells in ice-cold lysis buffer. Cell lysate proteins were then incubated with high capacity NeutrAvidin-agarose resin (Thermo Fisher, Waltham, MA, USA), and after washing in lysis buffer, bound proteins were eluted with SDS sample buffer and used in Western blot analyses as described above.
Western blot analysis
To validate NHERF-1 knock-down or overexpressing conditions in cultured astrocytes, protein of cell transfected with Nherf-1 shRNAs or GFP-NHERF-1 were separated by SDS–PAGE using 10% gels. The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-rad, Hercules, CA, USA). The blots were incubated overnight at 4 ℃ with anti-NHERF-1 (Thermo Fisher, Waltham, MA, USA, cat# PA1-090, 1:1,000) or anti-actin antibody (Sigma Aldrich, St. Louis, MO, USA, cat# A2066, 1:1,000). Blots were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA, 1:3000) followed by washing and detection of immunoreactivity with enhanced chemiluminescence (Bio-rad, Hercules, CA, USA).
Reverse transcription (RT) – PCR and real time PCR
For NHERF-1 KO validation, total RNA was isolated from mouse brain using an RNA purification Kit (GeneAll, Seoul, Korea) according to the manufacturer’s instructions. RT was performed with 1 µg total RNA using a cDNA Synthesis Kit (Takara Bio Inc, Kusatsu, Japan cat# 639543), according to the manufacturer’s instructions. PCR was performed using rTaq 5x PCR premix (ELPIS-BIOTECH, Daejeon, Korea, cat# EBT-1013) under the following cycle conditions: Denaturation at 95°C for 20 s, annealing at 55°C for 20 s, and extension at 72°C for 20 s. This cycle was repeated a total of 35 times. The PCR products were separated by electrophoresis in a 2% agarose gel, and images were captured on a gel imaging system. qPCR was also performed with SYBR Green mix (Enzo Life Sciences, Farmingdale, NY, USA, cat# ENZ-NUC104-1000). Primers were used the following sense and antisense primers: for NHERF-1, forward (5’-AGATCTGCCTCCAGCGATAC-3’) and reverse (5’-TTCATTTTTCTTGCTCCAGTCC-3’), and for GAPDH, forward (5’-GTCTTCACCACCATGGAGAA-3’) and reverse (5’- GCATGGACTGTGGTCATGAG-3’). GAPDH was used as a reference gene. The 2-△△CT method was used to calculate fold changes in gene expression.
Yeast two-hybrid assay
The TREK-1 N or C-terminus was cloned into the GAL4 DNA binding domain (BD) and the full length of NHERF-1 or NHERF-1 mutants (NHERF-1 PDZ1, PDZ2, C tail, NHERF-1 ΔPDZ1, ΔPDZ2 and ΔC tail) was cloned into the activation domain (AD). Direct interactions of the two proteins were investigated by co-transforming yeast AH190 cells with BD/TREK-1 N or C-terminus and AD/NHERF-1 WT or mutants. Transformed cells were then plated on synthetic dropout medium lacking Trp and Leu (TL-) at 30 ℃ for 3 days and were subsequently transferred to the medium lacking Trp, Leu, and His (TLH-) for growth selection.
Bimolecular fluorescence complementation (BiFC) assay
For BiFC, TREK-1, TWIK-1 and NHERF-1 were cloned into bimolecular fluorescence complement (pBiFC)-VN173 and pBiFC-VC155 vectors. To confirm the expression of each BiFC vector, additional Flag and HA tags were inserted in the C-terminal region of both BiFC vectors. HEK293T cells were co-transfected with cloned BiFC vectors in all possible pairwise combinations. The next day, these cells were fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.3% Triton X-100 for 5 min. After blocking for 1 h in 5% BSA solution, the cells were incubated with mouse anti-HA (Cell Signaling, Danvers, MA, USA cat# 2367S, 1: 250) and rabbit anti-Flag (Cell Signaling, Danvers, MA, USA cat# 14793S, 1:250) antibodies at 4°C overnight. Then, the cells were incubated with Alexa Fluor 594- or 647-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA, 1:400) for 1 h and then stained with DAPI to visualize nuclei. All images were acquired by confocal microscopy on a Nikon A1 confocal microscope.
Bioluminescence Resonance Energy Transfer (BRET) Assay
Fot BRET, TREK-1, TWIK-1 and NHERF-1 were cloned into mCit-PA-pBRET and NL-myc-pBRET vectors. HEK293T cells were cultured in 96-well plate (SPL) at a density of 1.5 ~ 2 x 10^5 cells per well. 24h after incubation, plasmids encoding donor and acceptor proteins are transfected at a 1:10 (total 500ng of DNA) with lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA). Coelenterazine-h (Sigma Aldrich, St. Louis, MO, USA, cat# c3230-50ug) was added to a final concentration of 5 µM per well (the total volume of 50 µl per well), and incubated for an additional 15 min. An infinite M200 Pro microplate reader (TECAN, Männedorf, Switzerland) was used with an integration time of 5 s to measure the short and long wavelengths with BLUE1 (370 to 480 nm) and GREEN1 (520 to 570 nm) filters for 1000 ms. BRET ratio was calculated as: (long-wavelength emission/short-wavelength emission) - (long-wavelength emission for donor (NL) only transfected cells/short-wavelength emission for donor (NL) only transfected cells).
Duolink proximity ligation assay (PLA)
Interactions between endogenous proteins were detected using a Duolink PLA kit (Sigma Aldrich, St. Louis, MO, USA, cat# DUO92014), according to the manufacturer’s instructions. The primary antibodies used for this assay were anti-TREK-1 (Santa Cruz Biotechnology, Dallas, TX, USA cat# sc-398449, 1:50) and anti-NHERF-1 (Bethyl Laboratories, Montgomery, TX, USA, cat# A302-974A, 1:100) antibodies. The PLA probe anti-rabbit minus binds to the anti-NHERF-1 antibody, whereas the PLA probe anti-mouse plus binds to the anti-TREK-1. Astrocytes growing on PDL-coated coverslips were observed using a Nikon Ti2 confocal microscope (Nikon Instruments Inc., Melville, NY, USA).
Immunohistochemistry
Virus infected mice were anesthetized using avertin and subjected to intracardiac perfusion with saline, followed by 4% PFA solution in PBS. Brains were fixed in 4% PFA overnight at 4 ℃, and then 40 µm-thick sections were obtained using vibratome (Leica, Wetzlar, Germany, VT1200). The slices were permeabilized with 0.5% Triton X-100 in PBS for 30 min at room temperature and subsequently incubated in blocking buffer (5% normal donkey serum, 3% BSA, and 0.2% Triton X-100 in PBS) for 2 h at room temperature. The slices were then incubated with primary antibodies, Chicken anti-GFAP antibody (Thermo Fisher, Waltham, MA, USA, cat# PA1-10004, 1:500), Chicken anti-MAP2 antibody (Thermo Fisher, Waltham, MA, USA, cat# PA1-10005, 1:500), Rabbit anti-NeuN antibody (Thermo Fisher, Waltham, MA, USA, cat# 711054, 1:500) overnight at 4°C. The sections were washed thrice in PBS and incubated with suitable fluorescence Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA; 1:400). The tissue sections were counter stained with DAPI and were mounted on glass slides for microscopy. All images were acquired using a Nikon A1 confocal microscope (Nikon Instruments Inc, Melville, NY, USA).
Electrophysiological recording in cultured astrocytes
Cultured astrocytes were plated onto coverslips for electrophysiological experiments. The standard solution for the pipette contained 150 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (pH 7.2, adjusted with KOH). Standard bath solution contained in mM: 150 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 5.5 D-glucose, and 20 sucrose (pH 7.4, adjusted with NaOH). Patch pipettes were made from borosilicate glass capillaries (Warner Instruments, Washington, DC, USA). The pipette resistance was 5–6 MΩ. Whole-cell currents were recorded using a patch clamp amplifier (Axopatch 700B, Axon Instruments, Union City, CA, USA). Current–voltage relations were measured by applying ramped pulses (from − 150 mV to + 50 mV over 1000-ms) from a holding potential of -60 mV. A Digidata 1550 A interface (Axon Instruments, Union City, CA, USA) was used to convert digital– analog signals between the amplifier and computer. Data were sampled at 5 kHz and filtered at 1 kHz. Currents were analyzed with Clampfit software (Axon Instruments, Union City, CA, USA). All experiments were conducted at room temperature.
Electrophysiological recording in hippocampal slices
pAAV-GFAPp-mCherry, pAAV-GFAPp-GFP, pAAV-GFAPp-Nherf-1-2A-mCherry or pAAV-GFAPp-TREK-1 N4-2A-GFP virus injected brain slices (300 µm) containing the hippocampus were prepared using a vibrating blade microtome (Leica, Wetzlar, Germany, VT1200) in ice-cold oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) 130 NaCl, 2.5 KCl, 1.25 KH2PO4, 3 MgCl2, 1 CaCl2, 26 NaHCO3, and 10 D-glucose. Individual hippocampal slices were transferred to a recording chamber, which was constantly perfused with ACSF recording solution containing (in mM) 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1.5 CaCl2, 1.5 MgCl2, and 10 glucose saturated with 95% O2–5% CO2 at pH 7.4. The slices were recovered at room temperature for at least 1 h and electrophysiological recording. Patch pipettes had a resistance of 3–5 MΩ when filled with pipette solution containing (in mM) 140 KCl, 10 HEPES, 5 EGTA, 2 Mg-ATP, and 0.2 Na-GTP, adjusted to pH 7.4 with KOH. Whole-cell patch recordings were performed on hippocampal astrocytes with a voltage-clamp configuration using an Axopatch 700B (Axon Instruments, San Jose, CA, USA).
Stereotaxic injection
C57BL/6N mice (7–8 weeks old) were anaesthetized with avertin (2,2,2-tribromethanol in 2-methyl 2-butanol) and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA). Briefly, the scale was opened and two holes were drilled in the skull (-1.7 mm AP, ± 1.4 mm ML from bregma). pAAV-GFAPp-mCherry, pAAV-GFAPp-Nherf-1-2A-mCherry and pAAV-GFAPp-TREK-1 N4-2A-GFP virus (2.21 x 10^14 GC/ml) were packaged in the serotype DJ at KIST Virus Facility. These viruses were bilaterally injected (250 nL per side) into the hippocampal CA1 stratum radiatum (SR) area (1.55 − 1.6 mm DV from the dura) through a Hamilton Syringe with a syringe pump (KD Scientific, Holliston, MA, USA) that infused the virus at a speed of 0.1 µL/min. The Hamilton Syringe was left undisturbed at the injected points for 10 min.
Kainic acid-induced seizure behaviors
Kainic acid (Sigma Aldrich, St. Louis, MO, USA.) dissolved in saline was administered intraperitoneal (i.p) injection at a dose of 35 mg/kg. Animals were monitored for 90 min after the injection. Seizure scores were monitored every 5 min using a Racine scale (0, normal behavior; 1, immobilization; 2, head nodding; 3, whole body myoclonus; 4, continuous rearing and falling; 5, clonic–tonic seizure; 6, death 21. Non-responsive animals with a seizure score of 0 over 90 min were excluded from analysis.
Statistical analysis
All data are presented as means ± standard error of the mean (SEM). The significance of data for comparison was assessed by Student’s t-test (paired t-test or unpaired t-test) or one-way ANOVA followed by Turkey’s post hoc test test and significance levels are given as: n.s: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Prism9.0 software (GraphPad Software, San Diego, CA, USA) was used for carrying out the statistical analysis.