Astrocytic NHERF-1 increases seizure susceptibility by inhibiting surface expression of TREK-1

Astrocytic NHERF-1 increases seizure susceptibility by inhibiting surface expression of TREK-1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Astrocytic NHERF-1 increases seizure susceptibility by inhibiting surface expression of TREK-1 Eun Mi Hwang, Yeonju Bae, Ajung Kim, Shinae Lee, kim seongseop, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3974699/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Mature hippocampal astrocytes exhibit a linear current-to-voltage (I-V) K + membrane conductance, which is called passive conductance. It is estimated to enable astrocytes to keep potassium homeostasis in the brain. We previously reported that the TWIK-1/TREK-1 heterodimeric channels are crucial for astrocytic passive conductance. However, the regulatory mechanism of these channels by other binding proteins still remains elusive. Here, we identified Na+/H + exchange regulator-1 (NHERF-1), a protein highly expressed in astrocytes, as a candidate interaction partner for these channels. NHERF-1 endogenously bound to TWIK-1/TREK-1 in hippocampal cultured astrocytes. When NHERF-1 is overexpressed or silenced, surface expression and activity of TWIK-1/TREK-1 heterodimeric channels were inhibited or enhanced, respectively. Furthermore, we confirmed that reduced astrocytic passive conductance by NHERF-1 overexpressing in the hippocampus increases kainic acid (KA)-induced seizure sensitivity. Taken together, these results suggest that NHERF-1 is a key regulator of TWIK-1/TREK-1 heterodimeric channels in astrocytes and suppression of TREK-1 surface expression by NHERF-1 increases KA-induced seizure susceptibility via reduction of astrocytic passive conductance. astrocytic passive conductance TREK-1 NHERF-1 kainic acid-induced seizures Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Astrocytes have been attributed to several functions in the CNS, including neurotransmitter clearance from synapses, direction during neuronal migration and active control of neuronal synaptic transmission. Additionally, astrocytes help to maintain the extracellular ionic environment required for communication between neurons 1 . In particular, the electrophysiological properties of astrocytes change during maturation processes. One of the prominent features in mature astrocytes are the more negative membrane potential and a linear I-V relationship. These features enable astrocytic ion homeostasis in the brain, such as extracellular K + buffering, called astrocytic passive conductance 2 – 4 . This may be suggested as a protective mechanism against the toxic accumulation of K + in the extracellular space and for modulating neuronal synchronization and network 3 , 5 . Previous studies have shown that TWIK-1 (K2P 1.1, KCNK1) and TREK-1 (K2P 2.1, KCNK2) channels, which belong to the two-pore domain K + channels (K2P) isoforms, form heterodimeric channels in a disulfide bond-dependent manner, and these channels predominantly mediate astrocytic passive conductance in the hippocampus 6 , 7 . Although our group has elucidated the molecular identity of adult astrocyte passive conductance 7 , 8 , its physiological or pathophysiological role is still poorly studied. To address these questions, we examined novel proteins that bind to the TWIK-1/TREK-1 heterodimer channel, targeting proteins abundantly expressed in adult astrocytes. NHERF-1 is a member of the PDZ (postsynaptic density protein 95/Drosophila disc large tumor suppressor/zonula occulens-1 (PSD-95/DIg/ZO-1)) family and scaffolding protein 9 , 10 . Scaffold proteins was functioned as crucial regulators of many signaling pathways through organize functional complexes organization and kinase activity modulation. They were characterized by the presence of protein–protein interaction modules including PDZ domains 11 . NHERF-1 contains two PDZ domains and one ezrin/radixin/moesin/merlin-binding domain (EBD) that attach to the cytoskeleton 12 . PDZ domains play an important role in regulating receptor and channel protein localization in several tissues and tight junctions and function to scaffold intracellular signaling protein complexes 10 . NHERF-1 mRNA is present in several regions of the mammalian brain 13 , 14 . In particular, NHERF-1 protein expression is higher in astrocytes than in neurons and interacts with TRPC4 channels and GLAST 15 , 16 . The lack of NHERF-1 in cultured astrocytes alters subcellular localization and the distribution of Group II metabotropic glutamate receptor 2 and 3 (mGluR2/3) through interactions 17 . Additionally, mutations and overexpression of NHERF-1 have been reported in several cancers, including glioblastoma 18 – 20 . In the present study, we found that the PDZ domains of NHERF-1 bind to the N-terminus of the TREK-1 channel. Specifically, NHERF-1 knockdown or overexpression in cultured astrocytes regulates the surface expression of TREK-1 and TREK-1-mediated channel activity. The heterogeneous NHERF-1 knockout (KO) mice had increased astrocyte passive conductance, whereas overexpression of NHERF-1 resulted in decreased astrocyte passive conductance and increased kainic acid (KA)-induced seizure susceptibility. Additionally, the reduced astrocyte passive conductance under NHERF-1 overexpression was completely rescued by overexpression of TREK-1 N4, the binding domain of TREK-1 for NHERF-1. Therefore, these data demonstrate that NHERF-1 plays an important role in the regulation of its surface expression, channel activity, and seizure susceptibility through interaction with TREK-1 in astrocytes. Materials and Methods 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. Results NHERF-1 inversely regulates K + currents in cultured astrocytes The PDZ domains play an important role in regulating receptor and channel protein localization in several tissues and tight junctions and function to scaffold intracellular signaling protein complexes 10 . We checked the expression level of Nherf-1 in the astrocytes and neurons using Brain-RNA seq database ( https://www.brainrnaseq.org , Figure S1 A). According to this database, the Nherf-1 gene was more than expressed in the astrocytes compared to neurons. We also confirmed the protein expression level of NHERF-1 in cultured astrocytes or neurons using a western blot. The NHERF-1 protein was more expressed in cultured astrocytes than cultured neurons (Figure S1 B) Therefore, NHERF-1 was highly expressed in the astrocytes of the brain. To assess the effect of NHERF-1 on K + channels in cultured astrocytes, we first recorded K + currents under the NHERF-1 overexpressed conditions. In previous studies, NHERF-1 regulated the activity or surface expression of several channels in various cells or tissues 22 – 24 . First, the NHERF-1 overexpressed condition showed the increased expression of NHERF-1 in cultured astrocytes (Fig. 1 A). K + currents in NHERF-1 overexpressed cultured astrocytes significantly decreased linearly (Figs. 1 B and C), and the reversal potential was depolarized from − 82.69 ± 1.58 mV to -69.88 ± 3.38 (Fig. 1 D). To test the opposite effect, we produced NHERF-1-specific short hairpin-forming interference RNA (shRNA). The expression level of NHERF-1 was effectively reduced by Nherf-1 shRNA in cultured astrocytes (Fig. 1 E). Next, we found that K + currents were significantly increased in cultured astrocytes by Nherf-1 shRNA (Figs. 1 F and G). The reversal potential was not changed (Fig. 1 H). These results suggest that NHERF-1 reversely regulates K + currents in cultured astrocytes. NHERF-1 mainly contributes to TWIK-1/TREK-1-mediated K + currents in cultured astrocytes. To identify the channels contributing to the K + current regulated by NHERF-1, we applied various K + channel inhibitors. K + channels are a superfamily of diverse members involved in K + currents in a variety of cell types, including astrocytes. Based on protein structure, K + channels are classified into the voltage-gated K + channels (Kv), inwardly rectifying K + channels (Kir), two-pore domain K + channels (K2P), and Ca 2+ -activated K + channels (KCa) families 4 , 25 – 27 . We added inhibitors for each of the four types of K + channels (TEA, Ba2+, apamin, and spadin) to cultured astrocytes. We only used astrocytes within 7 days of primary culture at P1, and as expected from the known expression level, we confirmed that spadin was most effectively inhibited (Figs. 2 A and B, Figures S2A and B). This was consistent with previous findings from our group 7 , 8 . Next, we investigated the effect of spadin on K + currents modulated by NHERF-1. K + currents were significantly reduced in GFP-NHERF-1 transfected cultured astrocytes compared to GFP control transfected cells. Interestingly, spadin had relatively little effect on K + currents in astrocytes overexpressing NHERF-1, but effectively reduced the current in control cells (Figs. 2 C and D). We measured spadin-sensitive K + currents in astrocytes by subtracting the pre-K + current from the K + current after treatment with spadin. Spadin-sensitive currents were smaller in GFP-NHERF-1 transfected astrocytes than in control cells (Figs. 2 E and F). Together, these data indicate that NHERF-1 is primarily involved in regulating TWIK-1/TREK-1 heterodimer channel-mediated K + currents in cultured astrocytes. Identification of binding regions between NHERF-1 and TREK-1 In a previous study, we confirmed that only the TWIK-1/TREK-1 heterodimer mainly exists in astrocytes 7 , so we tested whether NHERF-1 would interact with either TREK-1 or TWIK-1 channels. We applied bioluminescence resonance energy transfer (BRET) analysis to determine the interaction between the two proteins. The BRET signal ratio of TREK-1/NHERF-1 was significantly higher than that of TWIK-1/NHERF-1 (Figures S3A and B). These data suggest that NHERF-1 interacts better with TREK-1 than with TWIK-1. Next, to find the detailed binding site of TREK-1 on NHERF-1, we generated vector constructs expressing only the N- and C-terminal regions (TREK-1-N or TREK-1-C). The N and C termini of K2P channels are present in the cytoplasmic region of the cell 26 , 28 . Therefore, by performing a yeast two-hybrid assay, we confirmed that NHERF-1 is interacting with the N terminus of TREK-1 (Fig. 3 A). Co-IP experiments using the same strategy also clearly showed that NHERF-1 binds to the N terminus of TREK-1 (Fig. 3 B). To reconfirm the interaction between NHERF-1 and TREK-1-N, Bi-Fluorescence Complementation (BiFC) constructs expressing N- or C-terminal region deleted forms of TREK-1 (TREK-1-ΔN or TREK-1-ΔC) was produced (Fig. 3 C). Cells cotransfected with TREK-1/NHERF-1 and TREK-1-ΔC/NHERF-1 showed strong fluorescence, whereas cells cotransfected with TREK-1ΔN/NHERF-1 did not detect BiFC signal (Fig. 3 D). To further determine the binding site of NHERF-1 within TREK-1, the N terminus of TREK-1 was divided into four sections (ΔN1, ΔN1,2, ΔN1,2,3, or ΔN) (Figure S4A) 29 . BiFC signals were detected for TREK-1 ΔN1, TREK-1 ΔN1,2, and TREK-1 ΔN1,2,3 using NHERF-1, but not for TREK-1 ΔN using NHERF-1 (Figure S4B). NHERF-1 has been shown to interact with the N4 region of the N terminus of TREK-1. We therefore performed Co-IP and BiFC analyses using the N4 region deletion construct (TREK-1 ΔN4). As expected, TREK-1 ΔN4 showed low interaction across these assays (Figs. 3 E and F). These results indicate that NHERF-1 binds to the N4 region of TREK-1. To determine the binding region of NHERF-1 to TREK-1, we also generated several mutant constructs of NHERF-1 (Fig. 4 A). NHERF-1 contains two PDZ domains and one C-terminal ezrin-binding domain (EBD) 18 . We tested an interaction between TREK-1 and several domains of NHERF-1 by showing a positive yeast colony in the Y2H assay. The Y2H assay showed that TREK-1 binds to the PDZ1 or PDZ2 domain of NHERF-1 (Fig. 4 A). Co-IP data also showed that TREK-1 strongly interacts with the PDZ1 domain of NHERF-1 and weakly binds with PDZ2 domain (Fig. 4 B). To confirm the interaction between PDZ1 domain of NHERF-1 and TREK-1, we produced BiFC constructs expressing vector PDZ1, PDZ2 or C-terminal region deleted forms of NHERF-1 (NHERF-1 ΔPDZ1, NHERF-1 ΔPDZ2, NHERF-1 ΔC, NHERF-1 ΔPDZ1,2) (Fig. 4 C). We performed a BiFC assay to confirm these interactions. The BiFC data showed that the fluorescence signal was detected in cells co-transfected with TREK-1/NHERF-1, TREK-1/NHERF-1 ΔPDZ1, TREK-1/NHERF-1 ΔPDZ2 and TREK-1/NHERF-1 ΔC, whereas not detected in cells co-transfected with TREK-1/NHERF-1 ΔPDZ1 and 2 (Fig. 4 D). These results indicate that PDZ domains of NHERF-1 bind to the N4 region of TREK-1. NHERF-1 regulates surfaced expression of TREK-1 via their interaction in cultured astrocytes To identify whether NHERF-1 endogenously interacts with TREK-1 in astrocytes, we performed a Co-IP and Duolink proximity ligation assay (PLA). Co-immunoprecipitation (Co-IP) experiments showed an interaction between TREK-1 and NHERF-1 with anti-NHERF-1 antibody in cultured astrocytes (Fig. 5 A). This interaction was emphasized by the Duolink PLA signals. A strong Duolink PLA signal from TREK-1 and NHERF-1 was observed in astrocytes compared to only the exposed anti-TREK-1 antibody (Fig. 5 B). Channel activity is associated with surface expression in cells, and this concept commonly serves as a mechanism for regulating channel activity 30 , 31 . To determine the effect of NHERF-1 on the surface expression of the TREK-1 channel, we preferentially confirmed the surface expression of TREK-1 in NHERF-1 overexpressed or NHERF-1 knocking down cultured astrocytes. Cell surface biotinylation assay indicated that the surface expression of TREK-1 was markedly enhanced in the NHERF-1 knockdown condition using Nherf-1 shRNA in cultured astrocytes (Figs. 5 C and D). We confirm the reverse effect by testing it under the NHERF-1 overexpressed condition. NHERF-1 overexpression significantly reduced the surface expression of TREK-1 (Figs. 5 E and F). These results suggest that NHERF-1 inversely regulates cell surface expression of TREK-1 through physical interactions in cultured astrocytes. Astrocytic passive conductance is enhanced in heterozygous NHERF-1 knockout mice To clarify whether NHERF-1 mediates astrocytic passive conductance via TWIK-1/TREK-1 heterodimeric channels in vivo, we used the NHERF-1 gene knockout (KO) animal model. The genomic illustration of NHERF-1 KO mice showed that homologous recombination replaced the first exon containing the beginning of the transcription of the mouse Nherf-1 gene with the neomycin sequence (Figure S5A) 32 . To verify the genetic deletion of NHERF-1 mice, we performed PCR-based genotyping of tail samples from NHERF-1 wild-type (WT) and heterozygous (Het) mice (Figure S5B). qRT-PCR analysis revealed the mRNA expression levels of Nherf-1. In NHERF-1 Het mice, qRT-PCR results confirmed that Nherf-1 mRNA was reduced by almost half (Figures S5C and D). We also examined the expression level of NHERF-1 protein in brain tissue using a specific anti-NHERF-1 antibody (Figure S5E) To determine the contribution of NHERF-1 to TWIK-1/TREK-1 heterodimeric channel-mediated astrocytic passive conductance, we aimed to measure the effects of NHERF-1 on astrocytic passive conductance in hippocampal slices of NHERF-1 Het mice. The electrophysiological recording clearly showed that passive conductance in NHERF-1 Het mice was more enhanced than that in WT mice. The application of spadin was significantly modulated in both WT and Het mice (Fig. 6 A). The linear current-to-voltage (I-V) relationship of passive conductance reflects the intrinsic properties of mature astrocytes 6 – 8 . NHERF-1 Het mice also showed an increased linear I-V compared with WT mice (Figs. 6 B and C). The spadin-sensitive currents were consistent with these results in the NHERF-1 WT and Het astrocytes (Figs. 6 D and E). Together, these results suggest that NHERF-1 regulates TWIK-1/TREK-1 heterodimeric channels-mediated astrocytic passive conductance. Dysfunction of passive conductance by astrocyte-specific NHERF-1 overexpression accelerates seizure susceptibility Among the previous reports, the expression of NHERF-1 is exhibited in various cells of the brain such as ependymal epitheliums 12 . Because the NHERF-1 KO can't exclude the effects of another cell type, we injected the NHERF-1 overexpressing virus using astrocyte-specific truncated GFAP (GfaABC1D) promoter into the hippocampal CA1 stratum radiatum (SR) regions for 2 weeks (Fig. 7 A). The expression of mCherry protein was observed only in astrocytes, not neurons, of the hippocampal tissue infected with the AAV-GFAPp-NHERF-1-2A-mCherry virus (Fig. 7 B). Next, we investigated whether NHERF-1 regulated passive conductance in hippocampal astrocytes. The electrophysiological recording exhibited a huge passive conductance in astrocytes infected AAV-GFAPp-mCherry virus, but when NHERF-1 was overexpressed by AAV-GFAPp-NHERF-1-2A-mCherry virus, passive conductance was dramatically decreased. The reduction of astrocytic passive conductance by NHERF-1 overexpression had no effect after Spadin treatment (Figs. 7 C-E). The spadin-sensitive currents were coherent with these results (Figs. 7 F and G). Dysregulation of extracellular K + concentration via astrocytic potassium buffering involved in epileptic seizure 33 , 34 . To identify the seizure sensitivity when NHERF-1 overexpressed in astrocytes, which showed decreased astrocytic passive conductance, we observed seizure behavior following the intraperitoneally (i.p) injection of 35 mg/kg kainic acid (KA) in the AAV-GFAPp-mCherry or AAV-GFAPp-NHERF-1-2A-mCherry virus-infected mice (Fig. 7 H). The behavioral seizure responses were monitored using a modified Racine score for 90 minutes after the KA injection 21 . KA-induced seizure responses were bigger in AAV-GFAPp-NHERF-1-2A-mCherry virus-infected mice than in control mice during 90 mins (Fig. 7 I). We also found that the cumulative score of NHERF-1 overexpressed mice was higher than control mice. But latency to score 3 was not altered in between both mice. (Figs. 7 J and K). These data suggested that When NHERF-1 is overexpressed only in astrocytes, the seizure response is severe because of the reduction of astrocytic passive conductance. Additional overexpression of TREK-1 N4 effectively reverses the effects of NHERF-1 overexpression Next, we asked whether TREK-1 N4, the interaction region of TREK-1 and NHERF-1, prevented the dysfunction of passive conductance and KA-induced seizure susceptibility in NHERF-1 overexpression mice. Before the in vivo experiments, we confirmed the recovery ability by TREK-1 N4 of K + currents under the cultured system. K + currents of mCherry-NHERF-1 transfected astrocytes were decreased, whereas they recovered by HA-TREK-1-2A-GFP overexpression (Figures S6A and B). Next, we generated TREK-1 N4 overexpression virus using GFAP (GfaABC1D) promoter and injected AAV-GFAPp-NHERF-1-2A-mCherry and/or AAV-GFAPp-TREK-1 N4 2A-GFP virus into the hippocampal CA1 stratum radiatum (SR) regions for 2 weeks (Fig. 8 A). The expression mCherry and GFP protein was observed only in astrocytes, not neurons, of the hippocampal tissue infected with the AAV-GFAPp-NHERF-1-2A-mCherry virus and/or AAV-GFAPp-TREK-1 N4-2A-GFP (Fig. 8 B, S7A and B). Same as the cultured system, reduced TWIK-1/TREK-1-mediated passive conductance in NHERF-1 astrocyte-specific overexpressed mice was rescued by AAV-GFAPp-TREK-1 N4-2A-GFP virus infection (Figs. 8 C-G). The KA-induced behavioral seizure responses during the 90 min observation indicated that AAV-GFAPp-NHERF-1-2A-mCherry and AAV-GFAPp-TREK-1 N4-2A-GFP-infected mice showed delayed seizure onset compared to AAV-GFAPp-NHERF-1-2A-mCherry only infected mice (Fig. 8 H and I). The cumulative score for 20 mins also decreased in AAV-GFAPp-NHERF-1-2A-mCherry and AAV-GFAPp-TREK-1 N4-2A-GFP-infected mice (Fig. 8 J). However, latency to score 3 was not changed between both mice (Fig. 8 K). These results highlight that the decreased potassium conductance of astrocytes and increased seizure susceptibility caused by NHERF-1 overexpression were restored by additional TREK-1 N4. Discussion In this study, we found that NHERF-1 is abundantly expressed in astrocytes and not only regulates intrinsic K + currents in cultured cells (Fig. 1 ) but also contributes to TWIK-1/TREK-1-mediated passive conductance in adults (Fig. 2 ). Interestingly, we discovered NHERF-1 as a novel binding protein for the TREK-1 channel and confirmed its protein-protein interaction in vitro (Figs. 3 and 4 ). Moreover, NEHRF-1 was associated with endogenous TREK-1 in cultured astrocytes, and overexpression or knockdown of NHERF-1 regulated the surface expression of TREK-1 (Fig. 5 ). Astrocyte passive conductance was increased in NHERF-1 het KO mice with reduced expression of NHERF-1 (Fig. 6 ), whereas NHERF-1 overexpression had the opposite effect. Astrocyte-selective overexpression of NHERF-1 reduced astrocyte passive conductance and predisposed KA-evoked seizure responses (Fig. 7 ). However, this phenomenon is significantly restored by TREK-1 N4, which interferes with the binding of NHERF-1 to TREK-1 (Fig. 8 ). The present study demonstrates that NHERF-1 can act as an effective regulator of TREK-1-mediated passive conductance in glial cells. Additionally, it provides evidence that changes in NHERF-1 expression can influence KA-induced seizure sensitivity through alterations in astrocytic passive conductance. One of the unique functions of astrocytes is the regulation of ionic homeostasis in the brain, such as extracellular K + buffering through K + channels. Astrocytes show a linear I-V relationship with a negative membrane potential (Vm), called passive conductance. This phenomenon results from leakage of K + membrane conductance, and it is not affected by time and voltage 3 , 4 , 35 . Although the exact molecular mechanism is not yet known, it appears to be partly due to weakly rectifying K + (Kir) channels and two-pore domain K + (K2P) channels, especially the TWIK-1/TREK-1 heterodimer channel 6 , 7 , 35 , 36 . These changes in ionic homeostasis can induce a variety of symptoms. Mice lacking Kir4.1 in astrocytes show no change in passive conductance but rapidly hyperpolarize the membrane potential of astrocytes and develop severe spontaneous seizures after birth 37 . However, TWIK-1/TREK-1 double KO mice not only did not show the expected changes in astrocytic passive conductance but also showed no pathological behavior 38 . Since this may be due to a compensatory effect during development, we thought that experiments using viruses in adults would be a more reliable alternative to confirm the role of effective passive conductance. In this study, we used an NHERF-1 overexpressing virus in adults and observed that it can regulate astrocytic passive conductance through interaction with TREK-1. NHERF-1 is a member of the PDZ family, which represents the most common protein-protein interaction domain 9 , 10 , 39 . Subcellular localization, endocytosis, transport, and signaling of many receptors and channels are regulated through interaction with PDZ domains in various cells or tissues 10 , 40 . We demonstrated that TREK-1 binds to the PDZ domain of NHERF-1 and that this interaction regulates spadin-sensitive astrocytic passive conductance (Fig. 8 ). These findings suggest the possibility that TREK-1 may interact with other families of proteins that possess PDZ domains in addition to NHERF-1. TREK-1 has a short N-terminus consisting of 46 amino acids and most of the reported TREK-1 binding proteins bind to its relatively long C-terminus. Interestingly, in this study, NHERF-1 was identified as the protein that binds to the N-terminus of TREK-1, next to β-COP. β-COP was identified as an interacting protein of TREK-1 in astrocytes, binding to the N-terminal domain and increasing both surface expression and channel activity 29 , 41 . However, NHERF-1 is bound to the N-terminal domain of TREK-1 but reduces both surface expression and channel activity. Because both of these proteins bind to the TREK-1 N-terminal domain, especially the N4 region, and induce opposite effects, it may be important which protein binds predominantly in a physiological condition. We observed no changes in K + currents when only N4 was expressed in cultured astrocytes (data not shown). If NHERF-1 expression is relatively increased in certain circumstances, such as cancer development, a decrease in TREK-1-mediated K + currents in astrocytes is expected (Fig. 7 ). It has been reported that many glioblastoma patients suffer from headaches and seizures 42 , 43 and one of the reasons for this may be the increased seizure susceptibility caused by NHERF-1 overexpression in astrocytes, according to our results. Further studies are needed to determine the causal relationship between increased expression of NHERF-1 and pathological conditions. NHERF-1 is widely distributed in the rodent brain and is abundantly expressed in astrocytes rather than neurons. This is crucial because astrocytes are a specific group of glial cells that have functions such as the control of ionic and osmotic composition of the extracellular environment. NHERF-1 in astrocytes supports a role for the PDZ domain as a possible regulator of membrane transporters and channels. In a previous report, excitatory amino acid transporter 1 (EAAT1), called GLAST, colocalized and bound with NHERF-1 in astrocytes. This suggests that NHERF-1 may regulate glutamate homeostasis through GLAST in astrocytes 16 . Additionally, glial fibrillary acidic protein (GFAP), a characteristic intermediate filament protein of astrocytes, stabilizes and maintains GLAST at the plasma membrane through a series of intermediate linking proteins, including NHERF-1 and ezrin 44 . Therefore, this report demonstrates a novel function for NHERF-1 in regulating TWIK-1/TREK-1-mediated astrocytic passive conductance. Despite the many roles of NHERF-1 in astrocytes, there are no reports of NHERF-1 being significantly involved in brain function, other than reports that NHERF-1 KO mice exhibit hydrocephalus 12 , 45 . In this respect, this report provides new compelling evidence on the function of NHERF-1 in the brain, especially in astrocytes. We also found that the reduced astrocyte passive conductance under NHERF-1 overexpression was restored by TREK-1 N4 addition, and KA-evoked seizure behavior was also significantly rescued (Fig. 8 ). These findings suggest that dysfunction of astrocyte passive conductance plays an important role in epileptic seizures and that dissociation of TREK-1 and NHERF-1 complexes should be considered a promising target for seizure therapy. This new target may resolve various side effects caused by existing antiseizure drugs targeting nerve cells. In conclusion, we have identified NHERF-1 as a novel binding protein for TWIK-1/TREK-1 channels and studied the regulatory mechanisms between them, resulting in a better understanding of the function of astrocytes. Additionally, we proposed a new therapeutic strategy targeting astrocytes for the treatment of seizures. Declarations Acknowledgments This work was supported by the National Research Foundation (NRF) of Korea (NRF2022R1A2C1093143), through its funding to J.Y.P. This research was also supported by a grant of NRF of Korea (2020R1A2C2010650) and KIST intramural grant (2E32901), awarded to E.M.H. Conflict of interests The authors declare no competing interests. Supplementary Information available online. Author Contributions G.Y., J.-Y.P. and E.M.H. designed this study, while J.-Y.P. and E.M.H. supervised and edited the manuscript. Y.B. conducted most of the experiments and acquired the data. A.K. injected AAV and performed behavioral experiments. S. L. conducted most co-IP experiments using overexpressed cells, and S.-S. K. performed co-IP experiments using TREK-1 Δ4 mutant overexpressed cells. K.R. conducted co-IP experiments using primary cultured astrocytes. S.P. performed binding analyses using a yeast two hybrid system Reviewer Information included if applicable References Purushotham, S. S. & Buskila, Y. Astrocytic modulation of neuronal signalling. Frontiers in Network Physiology 3 , 1205544 (2023). Kofuji, P. & Newman, E. Potassium buffering in the central nervous system. Neuroscience 129 , 1043-1054 (2004). Bellot-Saez, A., Kékesi, O., Morley, J. W. & Buskila, Y. Astrocytic modulation of neuronal excitability through K+ spatial buffering. Neuroscience & Biobehavioral Reviews 77 , 87-97 (2017). Ryoo, K. & Park, J.-Y. Two-pore domain potassium channels in astrocytes. Experimental neurobiology 25 , 222 (2016). Wang, F. et al. Astrocytes modulate neural network activity by Ca2+-dependent uptake of extracellular K+. Science signaling 5 , ra26-ra26 (2012). Zhou, M. et al. TWIK-1 and TREK-1 are potassium channels contributing significantly to astrocyte passive conductance in rat hippocampal slices. Journal of Neuroscience 29 , 8551-8564 (2009). Mi Hwang, E. et al. A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes. Nature communications 5 , 3227 (2014). Bae, Y. et al. Spadin modulates astrocytic passive conductance via inhibition of TWIK-1/TREK-1 heterodimeric channels. International journal of molecular sciences 21 , 9639 (2020). Bushau-Sprinkle, A. M. & Lederer, E. D. New roles of the Na+/H+ exchange regulatory factor 1 scaffolding protein: a review. American Journal of Physiology-Renal Physiology 318 , F804-F808 (2020). Dunn, H. A. & Ferguson, S. S. PDZ protein regulation of G protein–coupled receptor trafficking and signaling pathways. Molecular pharmacology 88 , 624-639 (2015). Vaquero, J., Nguyen Ho-Bouldoires, T., Claperon, A. & Fouassier, L. Role of the PDZ-scaffold protein NHERF1/EBP50 in cancer biology: from signaling regulation to clinical relevance. Oncogene 36 , 3067-3079 (2017). Treat, A. C. et al. The PDZ protein Na+/H+ exchanger regulatory factor-1 (NHERF1) regulates planar cell polarity and motile cilia organization. PloS one 11 , e0153144 (2016). Ediger, T. R., Kraus, W. L., Weinman, E. J. & Katzenellenbogen, B. S. Estrogen receptor regulation of the Na+/H+ exchanger regulatory factor. Endocrinology 140 , 2976-2982 (1999). Weinman, E. J., Steplock, D., Wang, Y. & Shenolikar, S. Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na (+)-H+ exchanger. The Journal of clinical investigation 95 , 2143-2149 (1995). Song, X. et al. Canonical transient receptor potential channel 4 (TRPC4) co‐localizes with the scaffolding protein ZO‐1 in human fetal astrocytes in culture. Glia 49 , 418-429 (2005). Lee, A. et al. Na+–H+ exchanger regulatory factor 1 is a PDZ scaffold for the astroglial glutamate transporter GLAST. Glia 55 , 119-129 (2007). Ritter-Makinson, S. L. et al. Group II metabotropic glutamate receptor interactions with NHERF scaffold proteins: Implications for receptor localization in brain. Neuroscience 353 , 58-75 (2017). Bhattacharya, S., Stanley, C. B., Heller, W. T., Friedman, P. A. & Bu, Z. Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly. Journal of Biological Chemistry 294 , 11297-11310 (2019). Shibata, T., Chuma, M., Kokubu, A., Sakamoto, M. & Hirohashi, S. EBP50, a β-catenin-associating protein, enhances Wnt signaling and is over-expressed in hepatocellular carcinoma. Hepatology 38 , 178-186 (2003). Kislin, K. L., McDonough, W. S., Eschbacher, J. M., Armstrong, B. A. & Berens, M. E. NHERF-1: modulator of glioblastoma cell migration and invasion. Neoplasia 11 , 377-IN377 (2009). Racine, R. J. Modification of seizure activity by electrical stimulation: cortical areas. Electroencephalography and clinical neurophysiology 38 , 1-12 (1975). Storch, U. et al. Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol. Proceedings of the National Academy of Sciences 114 , E37-E46 (2017). Martin, E. R., Barbieri, A., Ford, R. C. & Robinson, R. C. In vivo crystals reveal critical features of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and the PDZ2 domain of Na+/H+ exchange cofactor NHERF1. Journal of Biological Chemistry 295 , 4464-4476 (2020). Saha, T. et al. Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner. Biochemistry and Biophysics Reports 25 , 100912 (2021). Choe, S. Potassium channel structures. Nature Reviews Neuroscience 3 , 115-121 (2002). Enyedi, P. & Czirják, G. Molecular background of leak K+ currents: two-pore domain potassium channels. Physiological reviews 90 , 559-605 (2010). Coetzee, W. A. et al. Molecular diversity of K+ channels. Annals of the New York Academy of Sciences 868 , 233-255 (1999). Lesage, F. et al. TWIK‐1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure. The EMBO journal 15 , 1004-1011 (1996). Kim, S.-S. et al. β-COP Regulates TWIK1/TREK1 Heterodimeric Channel-Mediated Passive Conductance in Astrocytes. Cells 11 , 3322 (2022). Solé, L. et al. KCNE4 suppresses Kv1. 3 currents by modulating trafficking, surface expression and channel gating. Journal of cell science 122 , 3738-3748 (2009). Lee, Y.-S. et al. Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells. Scientific reports 6 , 26413 (2016). Shenolikar, S., Voltz, J., Minkoff, C., Wade, J. & Weinman, E. Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting. Proceedings of the National Academy of Sciences 99 , 11470-11475 (2002). Wang, F., Qi, X., Zhang, J. & Huang, J. H. Astrocytic modulation of potassium under seizures. Neural Regeneration Research 15 , 980 (2020). David, Y. et al. Astrocytic dysfunction in epileptogenesis: consequence of altered potassium and glutamate homeostasis? Journal of Neuroscience 29 , 10588-10599 (2009). Nwaobi, S. E., Cuddapah, V. A., Patterson, K. C., Randolph, A. C. & Olsen, M. L. The role of glial-specific Kir4. 1 in normal and pathological states of the CNS. Acta neuropathologica 132 , 1-21 (2016). Olsen, M. L. et al. New insights on astrocyte ion channels: critical for homeostasis and neuron-glia signaling. Journal of Neuroscience 35 , 13827-13835 (2015). Djukic, B., Casper, K. B., Philpot, B. D., Chin, L.-S. & McCarthy, K. D. Conditional knock-out of Kir4. 1 leads to glial membrane depolarization, inhibition of potassium and glutamate uptake, and enhanced short-term synaptic potentiation. Journal of Neuroscience 27 , 11354-11365 (2007). Du, Y. et al. Genetic deletion of TREK-1 or TWIK-1/TREK-1 potassium channels does not alter the basic electrophysiological properties of mature hippocampal astrocytes in situ. Frontiers in cellular neuroscience 10 , 13 (2016). Feng, W. & Zhang, M. Organization and dynamics of PDZ-domain-related supramodules in the postsynaptic density. Nature Reviews Neuroscience 10 , 87-99 (2009). Lee, H.-J. & Zheng, J. J. PDZ domains and their binding partners: structure, specificity, and modification. Cell communication and Signaling 8 , 1-18 (2010). Kim, E. et al. Enhancement of TREK1 channel surface expression by protein–protein interaction with β-COP. Biochemical and biophysical research communications 395 , 244-250 (2010). Pace, A. et al. End of life issues in brain tumor patients. Journal of neuro-oncology 91 , 39-43 (2009). Sizoo, E. M. et al. Symptoms and problems in the end-of-life phase of high-grade glioma patients. Neuro-oncology 12 , 1162-1166 (2010). Sullivan, S. M. et al. Cytoskeletal anchoring of GLAST determines susceptibility to brain damage: an identified role for GFAP. Journal of Biological Chemistry 282 , 29414-29423 (2007). Georgescu, M.-M. et al. NHERF1/EBP50 is an organizer of polarity structures and a diagnostic marker in ependymoma. Acta neuropathologica communications 3 , 1-10 (2015). Additional Declarations (Not answered) Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3974699","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":276198894,"identity":"7efba65c-b878-4d8b-8162-e0a0be1c4eef","order_by":0,"name":"Eun Mi Hwang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYFACHjApJ8EM5UsQq8UYqIWxgSQtiTMYiNXCL3b2mMTPHbXpM9vZnz9g3GPDIDn7AH4tkrPz0iR7zxzPnc3MY9jA8CyNQZovAb8Wg9s5Zjd4247lzmPmATrswGEGOR4CDrMHarn5t+1Yuhwz+0Oglv+EtRhI55jd5m2rSZBmZgA67MABBmlCWiRu55j/lm07YDizmcdwRsKBZB7JHgJa+GfnGBu+bauTlzh//MGHDwfs5CTOENACBYchVAIsmogAdcQqHAWjYBSMgpEIAGmDPYaYJC/lAAAAAElFTkSuQmCC","orcid":"","institution":"Brain Science Institutue, Korea Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Eun","middleName":"Mi","lastName":"Hwang","suffix":""},{"id":276198895,"identity":"140c0b49-1501-4d85-8bdf-3f8a1acbb6e0","order_by":1,"name":"Yeonju Bae","email":"","orcid":"","institution":"Brain Science Institutue, Korea Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yeonju","middleName":"","lastName":"Bae","suffix":""},{"id":276198896,"identity":"ed63e6ca-3b59-4ec0-8e11-2eeb60acf792","order_by":2,"name":"Ajung Kim","email":"","orcid":"","institution":"Brain Science Institutue, Korea Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ajung","middleName":"","lastName":"Kim","suffix":""},{"id":276198897,"identity":"7714fb17-5192-4811-939f-20b729e49de6","order_by":3,"name":"Shinae Lee","email":"","orcid":"","institution":"Brain Science Institutue, Korea Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shinae","middleName":"","lastName":"Lee","suffix":""},{"id":276198898,"identity":"c2d250d1-4f71-43f1-874f-b5c0625bfba4","order_by":4,"name":"kim seongseop","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"kim","middleName":"","lastName":"seongseop","suffix":""},{"id":276198899,"identity":"2b33c6fb-946e-47a0-ab64-a6ffcadf6432","order_by":5,"name":"Sunyoung Park","email":"","orcid":"","institution":"School of Biosystems and Biomedical Sciences, College of Health Sciences, Korea University","correspondingAuthor":false,"prefix":"","firstName":"Sunyoung","middleName":"","lastName":"Park","suffix":""},{"id":276198900,"identity":"b663e0a7-33df-4dcd-82ea-0d3a9386c091","order_by":6,"name":"Kanghyun Ryoo","email":"","orcid":"","institution":"School of Biosystems and Biomedical Sciences, College of Health Sciences, Korea University","correspondingAuthor":false,"prefix":"","firstName":"Kanghyun","middleName":"","lastName":"Ryoo","suffix":""},{"id":276198901,"identity":"181f96a2-4b2f-4dc2-bd05-7e4e00578425","order_by":7,"name":"Gwan-Su Yi","email":"","orcid":"","institution":"Department of Bio and Brain Engineering, KAIST","correspondingAuthor":false,"prefix":"","firstName":"Gwan-Su","middleName":"","lastName":"Yi","suffix":""},{"id":276198902,"identity":"99ea9b07-1523-4a7b-bf49-9b79cad89df9","order_by":8,"name":"Jae-Yong Park","email":"","orcid":"https://orcid.org/0000-0001-7698-7890","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Jae-Yong","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2024-02-21 06:05:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3974699/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3974699/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52078052,"identity":"27ffb315-cb00-42cb-80a0-d30e9b0938f4","added_by":"auto","created_at":"2024-03-06 10:32:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":276843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHERF-1 inversely regulates potassium currents in cultured astrocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Top; Western blotting was performed to verify Nherf-1 shRNA in astrocytes. Bottom; A bar graph showing the relative protein expression level of NHERF-1 in the cultured astrocytes (n=3). B. Current densities showing to scrambled shRNA (Sc shRNA) or Nherf-1 shRNA transfected astrocytes. C. Summary bar graph of Sc shRNA or Nherf-1 shRNA currents at +50 mV and -150 mV as in B. D. Reversal potential (Vrev) in Sc shRNA or Nherf-1 shRNA transfected astrocytes. E. Top; Western blotting was performed to verify NHERF-1 overexpressed condition in astrocytes. Bottom; A bar graph showing the relative protein expression level of NHERF-1 in the cultured astrocytes (n=4). F. Current densities showing to GFP-control (GFP) or GFP-NHERF-1 transfected astrocytes. G. Summary bar graph of GFP or GFP-NHERF-1 currents at +50 and -150 mV as in F. H. Reversal potential (Vrev) in GFP-control (GFP) or GFP-NHERF-1 transfected astrocytes. The number on each bar indicates n for each condition. All values are mean±SEM. P-values were obtained using Student’s t-test. n.s: not significant, *p\u0026lt;0.05, **p \u0026lt; 0.01 and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/0f4eb221d7688326699656e0.png"},{"id":52078049,"identity":"f928d35d-97e9-4b1a-87ff-5526ca187304","added_by":"auto","created_at":"2024-03-06 10:32:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":263702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHERF-1 primarily contributes to TWIK-1/TREK-1-mediated K+ currents in cultured astrocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Normalized traces of various K+ channel inhibitors-sensitive currents of cultured astrocytes. B. The bar graph shows normalized various K+ channel inhibitors-sensitive currents densities at +50 mV and -150 mV. C. Current densities showing before and after application of spadin to GFP control (GFP) or GFP-NHERF-1 transfected astrocytes. D. Summary bar graph of C at +50 mV and -150 mV. E. Spadin-sensitive currents were calculated by difference between currents before and after 10 μM spadin application in C. F. Summary bar graph of GFP control (GFP/spadin sensitive) or GFP-NHERF-1 (GFP-NHERF-1/spadin sensitive) spadin-subtracted currents plotted at +50 mV and -150 mV. The number on each bar indicates n for each condition. All values are mean±SEM. P-values were obtained using one-way ANOVA followed by Bonferroni‘s post hoc test or Student’s t-test. n.s: not significant, *p\u0026lt;0.05, ** p \u0026lt; 0.01 and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/bb6ce1944d183b482229c066.png"},{"id":52078053,"identity":"d4cb3e5b-f5f5-48d0-a9b6-92d19f338858","added_by":"auto","created_at":"2024-03-06 10:32:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":731927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHERF-1 directly binds with N4 of N-terminus of TREK-1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Top; Diagram of the full-length (FL), N-, C-terminus of TREK-1. Bottom; Yeast two-hybrid assays show direct binding between NHERF-1 and N-terminal of TREK-1. B. Co-IP experiments suggest that NHERF-1 interacts with N-terminal of TREK-1. C. Diagram of the TREK-1 FL and TREK-1 N (ΔN) or C (ΔC) terminus deletion mutants and schematic diagram depicting the Bimolecular fluorescence complementation (BiFC). D. BiFC experiments were performed in HEK293T cells transfected with VC-NHERF-1 and VN-TREK-1, VN-TREK-1 ΔN or VN-TREK-1 ΔC. Scale bar, 10 μm. Data obtained from three independent experiments. The number on each bar indicates n for each condition. E. Top; Diagram of the TREK-1 FL or TREK-1 ΔN4. Bottom; BiFC experiments with VC-NHERF-1 and VN-TREK-1 or VN-TREK-1 ΔN4. Scale bar, 10 μm. F. Co-IP experiments to assess the interaction between NHERF-1 and TREK-1 FL or TREK-1 ΔN4.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/116023b9b6fff3168c3d25c4.png"},{"id":52078055,"identity":"7a3bae81-8a65-42ca-9361-6eebb4c9494f","added_by":"auto","created_at":"2024-03-06 10:32:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":707089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTREK-1 directly binds with PDZ domains of NHERF-1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Top; Diagram of the full length (FL) and PDZ1, PDZ2, or C tail of NHERF-1. Bottom; Yeast two-hybrid assays show interactions between TREK-1 and FL, PDZ1, PDZ2, or C tail of NHERF-1. B. Co-IP data show also interactions between TREK-1 and FL, PDZ1, PDZ2, or C tail of NHERF-1. C. Diagram of the FL of NHERF-1 and various deletion mutants of NHERF-1. D. BiFC experiments between VN-TREK-1 and VC-NHERF-1 or VC-NHERF-1 deletion mutants. Scale bar, 10 μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/b84f15072d211d429c8724d1.png"},{"id":52078048,"identity":"bab248ef-52b4-4040-83d2-bf35ea8f9802","added_by":"auto","created_at":"2024-03-06 10:32:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":559394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHERF-1 regulates the surface expression of TREK-1 in astrocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Co-immunoprecipitation (Co-IP) data shows interactions between TREK-1 and NHERF-1 in astrocytes. Whole-cell lysates were immunoprecipitated using anti-NHERF-1 antibody and then blotted using anti-TREK-1 antibody. B. Left; Representative images of Duolink PLA assays in astrocytes. Scale bar, 20 μm. Right; PLA signals were counted using ImageJ software and average numbers of spots per cell are presented in the graph. C. Cell surface biotinylation results from astrocytes transfected with scrambled shRNA (Sc shRNA) or Nherf-1 shRNA. D. The summary bar graph shows the summary of C. E. Cell surface biotinylation experiments with astrocytes transfected with GFP control (GFP) or GFP-NHERF-1. F. Data from three independent experiments are presented in the normalized bar graph as in E. Data obtained from three independent experiments. The number on each bar indicates n for each condition. All values are mean±SEM. P-values were obtained using Student’s t-test. *p\u0026lt;0.05 and ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/731412fe58859a0bf33db7ad.png"},{"id":52078054,"identity":"b2931255-0cb4-42a4-8997-55996607b7a1","added_by":"auto","created_at":"2024-03-06 10:32:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":320714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterozygous NHERF-1 KO mice showed an enhanced TREK-1-mediated astrocytic passive conductance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative traces of passive conductance induced by voltage stepping from -160 mV to +40 mV in hippocampal astrocytes from NHERF-1 WT or Het mice treated with or without 10 μM spadin (SP). B. I-V curves of passive conductance in A. C. Bar graphs showing averaged currents at -160 mV and +40 mV. D. SP-sensitive currents were calculated by difference between currents before and after SP application in B. E. Summary bar graph of SP-subtracted currents plotted at -160 mV and +40 mV (N=4, respectively). All values are mean±SEM. P-values were obtained with one-way ANOVA followed by Bonferroni‘s post hoc test or Student’s t-test. n.s: not significant, *p\u0026lt;0.05, **p \u0026lt; 0.01 and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/050b14dc61dd68bc2e2b6c2e.png"},{"id":52078050,"identity":"5663d90c-0334-4567-9155-ba51ef112718","added_by":"auto","created_at":"2024-03-06 10:32:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1319798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDysfunction of passive conductance by astrocyte NHERF-1 overexpression accelerates seizure susceptibility.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Diagram showing injection locations of AAV-GFAPp-mCherry or AAV-GFAPp-NHERF-1-2A-mCherry virus in hippocampal CA1 stratum radiatum (SR). B. Left; Representative fluorescence images of dual immunostaining of MAP2 (green), pAAV-GFAPp-NHERF-1-2A-mCh virus (red) and GFAP (purple) with DAPI staining of nuclei (blue) in mouse hippocampus. Scale bar, 100 μm. Right; Enlarged images of B. Scale bar, 50 μm. C. Representative traces of passive conductance induced by voltage stepping from -160 mV to +40 mV in AAV-GFAPp-mCherry or AAV-GFAPp-NHERF-1-2A-mCherry virus expressing astrocytes of SR with or without 10 μM spadin (SP) respectively. D. I-V curves of passive conductance in C. E. Bar graphs showing averaged currents at -160 mV and +40 mV. F. SP-sensitive currents were calculated by difference between currents before and after spadin application in D. G. Summary bar graph of SP-subtracted currents plotted at -160 mV and +40 mV (N=3 or 4, respectively). H. Schematic representation of the experimental timeline. Each virus was injected into mice, and 2 weeks later, an additional injection of 35 mg/kg kainic acid (KA) was administered intraperitoneally (i.p.) into the abdominal cavity. I. Time course of mean behavioral seizure score following KA injection. J. Total cumulative seizure score for each group. K. Latency to score 3 seizure for each group. The number on each bar indicates n for each condition. All values are mean±SEM. P-values were obtained with one-way ANOVA followed by Bonferroni‘s post hoc test or Student’s t-test. n.s: not significant, *p\u0026lt;0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 and ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/9f735fab0417994e53c5a9c5.png"},{"id":52078057,"identity":"356cd300-cfcb-4450-a42e-e8e22a5dbd73","added_by":"auto","created_at":"2024-03-06 10:32:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1367353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHERF-1 overexpression-mediated astrocytes dysfunction and seizure behavior were regained by TREK-1 N4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Diagram showing injection locations of AAV-GFAPp-NHERF-1-2A-mCherry and/or AAV-GFAPp-TREK-1 N4-2A-GFP virus in hippocampal CA1 stratum radiatum (SR). B. Left; Representative fluorescence images of dual immunostaining of pAAV-GFAPp-TREK-1 N4-2A-GFP (green), pAAV-GFAPp-NHERF-1-2A-mCh virus (red) and GFAP (purple) with DAPI staining of nuclei (blue) in mouse hippocampus. Scale bar, 50 μm. Right; Enlarged images of B. Scale bar, 20 μm. C. Representative traces of passive conductance induced by voltage stepping from -160 mV to +40 mV in AAV-GFAPp-NHERF-1-2A-mCherry and/or AAV-GFAPp-TREK-1 N4-2A-GFP virus expressing astrocytes of SR with or without 10 μM spadin (SP) respectively. D. I-V curves of passive conductance in C. E. Bar graphs showing averaged currents at -160 mV and +40 mV. F. SP-sensitive currents were calculated by difference between currents before and after spadin application in D. G. Summary bar graph of SP-subtracted currents plotted at -160 mV and +40 mV (N=2 or 3, respectively). H. Schematic representation of the experimental timeline. Each virus was injected into mice, and 2 weeks later, an additional injection of 35 mg/kg kainic acid (KA) was administered intraperitoneally (i.p.) into the abdominal cavity. I. Time course of mean behavioral seizure score following KA injection. J. Cumulative seizure score over 20 minutes for each group. K. Latency to score 3 seizure for each group. The number on each bar indicates n for each condition. All values are mean±SEM. P-values were obtained with one-way ANOVA followed by Bonferroni‘s post hoc test. n.s: not significant, *p\u0026lt;0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 and ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/38c7ae891578b931e37277d0.png"},{"id":53526795,"identity":"84b8e778-1af7-44f2-ae05-1511a03d6455","added_by":"auto","created_at":"2024-03-27 05:14:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2536423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/ed63f1d4-aa7d-4c46-8f62-fd22dd78c4c9.pdf"},{"id":52078047,"identity":"e1e9a565-1ad6-471e-b114-68390a3f0c42","added_by":"auto","created_at":"2024-03-06 10:32:15","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1050608,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3974699/v1/2ca0c624b64a5264a5caeda7.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Astrocytic NHERF-1 increases seizure susceptibility by inhibiting surface expression of TREK-1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAstrocytes have been attributed to several functions in the CNS, including neurotransmitter clearance from synapses, direction during neuronal migration and active control of neuronal synaptic transmission. Additionally, astrocytes help to maintain the extracellular ionic environment required for communication between neurons \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In particular, the electrophysiological properties of astrocytes change during maturation processes. One of the prominent features in mature astrocytes are the more negative membrane potential and a linear I-V relationship. These features enable astrocytic ion homeostasis in the brain, such as extracellular K\u0026thinsp;+\u0026thinsp;buffering, called astrocytic passive conductance \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This may be suggested as a protective mechanism against the toxic accumulation of K\u0026thinsp;+\u0026thinsp;in the extracellular space and for modulating neuronal synchronization and network \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that TWIK-1 (K2P 1.1, KCNK1) and TREK-1 (K2P 2.1, KCNK2) channels, which belong to the two-pore domain K\u0026thinsp;+\u0026thinsp;channels (K2P) isoforms, form heterodimeric channels in a disulfide bond-dependent manner, and these channels predominantly mediate astrocytic passive conductance in the hippocampus \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Although our group has elucidated the molecular identity of adult astrocyte passive conductance \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, its physiological or pathophysiological role is still poorly studied. To address these questions, we examined novel proteins that bind to the TWIK-1/TREK-1 heterodimer channel, targeting proteins abundantly expressed in adult astrocytes.\u003c/p\u003e \u003cp\u003eNHERF-1 is a member of the PDZ (postsynaptic density protein 95/Drosophila disc large tumor suppressor/zonula occulens-1 (PSD-95/DIg/ZO-1)) family and scaffolding protein \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Scaffold proteins was functioned as crucial regulators of many signaling pathways through organize functional complexes organization and kinase activity modulation. They were characterized by the presence of protein\u0026ndash;protein interaction modules including PDZ domains \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. NHERF-1 contains two PDZ domains and one ezrin/radixin/moesin/merlin-binding domain (EBD) that attach to the cytoskeleton \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. PDZ domains play an important role in regulating receptor and channel protein localization in several tissues and tight junctions and function to scaffold intracellular signaling protein complexes \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. NHERF-1 mRNA is present in several regions of the mammalian brain \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In particular, NHERF-1 protein expression is higher in astrocytes than in neurons and interacts with TRPC4 channels and GLAST \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The lack of NHERF-1 in cultured astrocytes alters subcellular localization and the distribution of Group II metabotropic glutamate receptor 2 and 3 (mGluR2/3) through interactions \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Additionally, mutations and overexpression of NHERF-1 have been reported in several cancers, including glioblastoma \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the present study, we found that the PDZ domains of NHERF-1 bind to the N-terminus of the TREK-1 channel. Specifically, NHERF-1 knockdown or overexpression in cultured astrocytes regulates the surface expression of TREK-1 and TREK-1-mediated channel activity. The heterogeneous NHERF-1 knockout (KO) mice had increased astrocyte passive conductance, whereas overexpression of NHERF-1 resulted in decreased astrocyte passive conductance and increased kainic acid (KA)-induced seizure susceptibility. Additionally, the reduced astrocyte passive conductance under NHERF-1 overexpression was completely rescued by overexpression of TREK-1 N4, the binding domain of TREK-1 for NHERF-1. Therefore, these data demonstrate that NHERF-1 plays an important role in the regulation of its surface expression, channel activity, and seizure susceptibility through interaction with TREK-1 in astrocytes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eSpadin 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 \u0026minus;\u0026thinsp;20\u0026deg;C and diluted to the required concentrations in standard bath solution immediately prior to experimentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eTransgenic 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\u0026ndash;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 \u0026minus;\u0026thinsp;5\u0026prime;-GAGAAGGGTCCAAATGGCTA-3\u0026prime;; NHERF-1 WT reverse primer \u0026minus;\u0026thinsp;5\u0026prime;-TTCGGCCTCATTCTGGTC-3\u0026prime; and NHERF-1 KO reverse primer \u0026minus;\u0026thinsp;5\u0026prime;-CGCCTTCTTGACGAGTTCTT-3\u0026prime;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHEK293T cells culture and transfection\u003c/h2\u003e \u003cp\u003eHEK293T 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\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePrimary astrocyte culture and transfection\u003c/h2\u003e \u003cp\u003ePrimary astrocytes were maintained in DMEM supplemented with 10% fetal bovine serum, 10% horse serum, and penicillin\u0026ndash;streptomycin. Cultures were maintained at 37\u0026deg;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of expression vectors\u003c/h2\u003e \u003cp\u003ecDNAs 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of shRNAs\u003c/h2\u003e \u003cp\u003eFor gene silencing in cultured astrocyte, Trek-1 shRNA were constructed and validated as described previously \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The target region of Nherf-1 shRNA is 5\u0026rsquo;-AGCGATACCAGTGAGGAGCTAAAT-3\u0026rsquo;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of viral vectors\u003c/h2\u003e \u003cp\u003eFor 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP) and immunoblotting\u003c/h2\u003e \u003cp\u003eFor 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\u0026ndash;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\u0026ndash;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell surface biotinylation\u003c/h2\u003e \u003cp\u003eBiotinylation assays were performed using Sulfo-NHS-SS-Biotin (Thermo Fisher, Waltham, MA, USA) according to the manufacturer\u0026rsquo;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eTo 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\u0026ndash;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eReverse transcription (RT) \u0026ndash; PCR and real time PCR\u003c/h2\u003e \u003cp\u003eFor NHERF-1 KO validation, total RNA was isolated from mouse brain using an RNA purification Kit (GeneAll, Seoul, Korea) according to the manufacturer\u0026rsquo;s instructions. RT was performed with 1 \u0026micro;g total RNA using a cDNA Synthesis Kit (Takara Bio Inc, Kusatsu, Japan cat# 639543), according to the manufacturer\u0026rsquo;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\u0026deg;C for 20 s, annealing at 55\u0026deg;C for 20 s, and extension at 72\u0026deg;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\u0026rsquo;-AGATCTGCCTCCAGCGATAC-3\u0026rsquo;) and reverse (5\u0026rsquo;-TTCATTTTTCTTGCTCCAGTCC-3\u0026rsquo;), and for GAPDH, forward (5\u0026rsquo;-GTCTTCACCACCATGGAGAA-3\u0026rsquo;) and reverse (5\u0026rsquo;- GCATGGACTGTGGTCATGAG-3\u0026rsquo;). GAPDH was used as a reference gene. The 2-△△CT method was used to calculate fold changes in gene expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid assay\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBimolecular fluorescence complementation (BiFC) assay\u003c/h2\u003e \u003cp\u003eFor 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\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBioluminescence Resonance Energy Transfer (BRET) Assay\u003c/h2\u003e \u003cp\u003eFot 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\u0026thinsp;~\u0026thinsp;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 \u0026micro;M per well (the total volume of 50 \u0026micro;l per well), and incubated for an additional 15 min. An infinite M200 Pro microplate reader (TECAN, M\u0026auml;nnedorf, 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDuolink proximity ligation assay (PLA)\u003c/h2\u003e \u003cp\u003e Interactions between endogenous proteins were detected using a Duolink PLA kit (Sigma Aldrich, St. Louis, MO, USA, cat# DUO92014), according to the manufacturer\u0026rsquo;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eVirus 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 \u0026micro;m-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\u0026deg;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eElectrophysiological recording in cultured astrocytes\u003c/h2\u003e \u003cp\u003eCultured 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\u0026ndash;6 MΩ. Whole-cell currents were recorded using a patch clamp amplifier (Axopatch 700B, Axon Instruments, Union City, CA, USA). Current\u0026ndash;voltage relations were measured by applying ramped pulses (from \u0026minus;\u0026thinsp;150 mV to +\u0026thinsp;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\u0026ndash; 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eElectrophysiological recording in hippocampal slices\u003c/h2\u003e \u003cp\u003epAAV-GFAPp-mCherry, pAAV-GFAPp-GFP, pAAV-GFAPp-Nherf-1-2A-mCherry or pAAV-GFAPp-TREK-1 N4-2A-GFP virus injected brain slices (300 \u0026micro;m) 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\u0026ndash;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\u0026ndash;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStereotaxic injection\u003c/h2\u003e \u003cp\u003eC57BL/6N mice (7\u0026ndash;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, \u0026plusmn; 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\u0026thinsp;\u0026minus;\u0026thinsp;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 \u0026micro;L/min. The Hamilton Syringe was left undisturbed at the injected points for 10 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eKainic acid-induced seizure behaviors\u003c/h2\u003e \u003cp\u003eKainic 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\u0026ndash;tonic seizure; 6, death \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Non-responsive animals with a seizure score of 0 over 90 min were excluded from analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The significance of data for comparison was assessed by Student\u0026rsquo;s t-test (paired t-test or unpaired t-test) or one-way ANOVA followed by Turkey\u0026rsquo;s post hoc test test and significance levels are given as: n.s: not significant, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Prism9.0 software (GraphPad Software, San Diego, CA, USA) was used for carrying out the statistical analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eNHERF-1 inversely regulates K\u0026thinsp;+\u0026thinsp;currents in cultured astrocytes\u003c/h2\u003e \u003cp\u003eThe PDZ domains play an important role in regulating receptor and channel protein localization in several tissues and tight junctions and function to scaffold intracellular signaling protein complexes \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We checked the expression level of Nherf-1 in the astrocytes and neurons using Brain-RNA seq database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.brainrnaseq.org\u003c/span\u003e\u003cspan address=\"https://www.brainrnaseq.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). According to this database, the Nherf-1 gene was more than expressed in the astrocytes compared to neurons. We also confirmed the protein expression level of NHERF-1 in cultured astrocytes or neurons using a western blot. The NHERF-1 protein was more expressed in cultured astrocytes than cultured neurons (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB) Therefore, NHERF-1 was highly expressed in the astrocytes of the brain.\u003c/p\u003e \u003cp\u003eTo assess the effect of NHERF-1 on K\u0026thinsp;+\u0026thinsp;channels in cultured astrocytes, we first recorded K\u0026thinsp;+\u0026thinsp;currents under the NHERF-1 overexpressed conditions. In previous studies, NHERF-1 regulated the activity or surface expression of several channels in various cells or tissues \u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. First, the NHERF-1 overexpressed condition showed the increased expression of NHERF-1 in cultured astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). K\u0026thinsp;+\u0026thinsp;currents in NHERF-1 overexpressed cultured astrocytes significantly decreased linearly (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C), and the reversal potential was depolarized from \u0026minus;\u0026thinsp;82.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58 mV to -69.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.38 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To test the opposite effect, we produced NHERF-1-specific short hairpin-forming interference RNA (shRNA). The expression level of NHERF-1 was effectively reduced by Nherf-1 shRNA in cultured astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Next, we found that K\u0026thinsp;+\u0026thinsp;currents were significantly increased in cultured astrocytes by Nherf-1 shRNA (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and G). The reversal potential was not changed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These results suggest that NHERF-1 reversely regulates K\u0026thinsp;+\u0026thinsp;currents in cultured astrocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNHERF-1 mainly contributes to TWIK-1/TREK-1-mediated K\u0026thinsp;+\u0026thinsp;currents in cultured astrocytes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo identify the channels contributing to the K\u0026thinsp;+\u0026thinsp;current regulated by NHERF-1, we applied various K\u0026thinsp;+\u0026thinsp;channel inhibitors. K\u0026thinsp;+\u0026thinsp;channels are a superfamily of diverse members involved in K\u0026thinsp;+\u0026thinsp;currents in a variety of cell types, including astrocytes. Based on protein structure, K\u0026thinsp;+\u0026thinsp;channels are classified into the voltage-gated K\u0026thinsp;+\u0026thinsp;channels (Kv), inwardly rectifying K\u0026thinsp;+\u0026thinsp;channels (Kir), two-pore domain K\u0026thinsp;+\u0026thinsp;channels (K2P), and Ca 2+ -activated K\u0026thinsp;+\u0026thinsp;channels (KCa) families \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. We added inhibitors for each of the four types of K\u0026thinsp;+\u0026thinsp;channels (TEA, Ba2+, apamin, and spadin) to cultured astrocytes. We only used astrocytes within 7 days of primary culture at P1, and as expected from the known expression level, we confirmed that spadin was most effectively inhibited (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B, Figures S2A and B). This was consistent with previous findings from our group \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Next, we investigated the effect of spadin on K\u0026thinsp;+\u0026thinsp;currents modulated by NHERF-1. K\u0026thinsp;+\u0026thinsp;currents were significantly reduced in GFP-NHERF-1 transfected cultured astrocytes compared to GFP control transfected cells. Interestingly, spadin had relatively little effect on K\u0026thinsp;+\u0026thinsp;currents in astrocytes overexpressing NHERF-1, but effectively reduced the current in control cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). We measured spadin-sensitive K\u0026thinsp;+\u0026thinsp;currents in astrocytes by subtracting the pre-K\u0026thinsp;+\u0026thinsp;current from the K\u0026thinsp;+\u0026thinsp;current after treatment with spadin. Spadin-sensitive currents were smaller in GFP-NHERF-1 transfected astrocytes than in control cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F). Together, these data indicate that NHERF-1 is primarily involved in regulating TWIK-1/TREK-1 heterodimer channel-mediated K\u0026thinsp;+\u0026thinsp;currents in cultured astrocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eIdentification of binding regions between NHERF-1 and TREK-1\u003c/h2\u003e \u003cp\u003eIn a previous study, we confirmed that only the TWIK-1/TREK-1 heterodimer mainly exists in astrocytes \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, so we tested whether NHERF-1 would interact with either TREK-1 or TWIK-1 channels. We applied bioluminescence resonance energy transfer (BRET) analysis to determine the interaction between the two proteins. The BRET signal ratio of TREK-1/NHERF-1 was significantly higher than that of TWIK-1/NHERF-1 (Figures S3A and B). These data suggest that NHERF-1 interacts better with TREK-1 than with TWIK-1.\u003c/p\u003e \u003cp\u003eNext, to find the detailed binding site of TREK-1 on NHERF-1, we generated vector constructs expressing only the N- and C-terminal regions (TREK-1-N or TREK-1-C). The N and C termini of K2P channels are present in the cytoplasmic region of the cell \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Therefore, by performing a yeast two-hybrid assay, we confirmed that NHERF-1 is interacting with the N terminus of TREK-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Co-IP experiments using the same strategy also clearly showed that NHERF-1 binds to the N terminus of TREK-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To reconfirm the interaction between NHERF-1 and TREK-1-N, Bi-Fluorescence Complementation (BiFC) constructs expressing N- or C-terminal region deleted forms of TREK-1 (TREK-1-ΔN or TREK-1-ΔC) was produced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Cells cotransfected with TREK-1/NHERF-1 and TREK-1-ΔC/NHERF-1 showed strong fluorescence, whereas cells cotransfected with TREK-1ΔN/NHERF-1 did not detect BiFC signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). To further determine the binding site of NHERF-1 within TREK-1, the N terminus of TREK-1 was divided into four sections (ΔN1, ΔN1,2, ΔN1,2,3, or ΔN) (Figure S4A) \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. BiFC signals were detected for TREK-1 ΔN1, TREK-1 ΔN1,2, and TREK-1 ΔN1,2,3 using NHERF-1, but not for TREK-1 ΔN using NHERF-1 (Figure S4B). NHERF-1 has been shown to interact with the N4 region of the N terminus of TREK-1. We therefore performed Co-IP and BiFC analyses using the N4 region deletion construct (TREK-1 ΔN4). As expected, TREK-1 ΔN4 showed low interaction across these assays (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). These results indicate that NHERF-1 binds to the N4 region of TREK-1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the binding region of NHERF-1 to TREK-1, we also generated several mutant constructs of NHERF-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). NHERF-1 contains two PDZ domains and one C-terminal ezrin-binding domain (EBD) \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. We tested an interaction between TREK-1 and several domains of NHERF-1 by showing a positive yeast colony in the Y2H assay. The Y2H assay showed that TREK-1 binds to the PDZ1 or PDZ2 domain of NHERF-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Co-IP data also showed that TREK-1 strongly interacts with the PDZ1 domain of NHERF-1 and weakly binds with PDZ2 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To confirm the interaction between PDZ1 domain of NHERF-1 and TREK-1, we produced BiFC constructs expressing vector PDZ1, PDZ2 or C-terminal region deleted forms of NHERF-1 (NHERF-1 ΔPDZ1, NHERF-1 ΔPDZ2, NHERF-1 ΔC, NHERF-1 ΔPDZ1,2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We performed a BiFC assay to confirm these interactions. The BiFC data showed that the fluorescence signal was detected in cells co-transfected with TREK-1/NHERF-1, TREK-1/NHERF-1 ΔPDZ1, TREK-1/NHERF-1 ΔPDZ2 and TREK-1/NHERF-1 ΔC, whereas not detected in cells co-transfected with TREK-1/NHERF-1 ΔPDZ1 and 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results indicate that PDZ domains of NHERF-1 bind to the N4 region of TREK-1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eNHERF-1 regulates surfaced expression of TREK-1 via their interaction in cultured astrocytes\u003c/h2\u003e \u003cp\u003eTo identify whether NHERF-1 endogenously interacts with TREK-1 in astrocytes, we performed a Co-IP and Duolink proximity ligation assay (PLA). Co-immunoprecipitation (Co-IP) experiments showed an interaction between TREK-1 and NHERF-1 with anti-NHERF-1 antibody in cultured astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This interaction was emphasized by the Duolink PLA signals. A strong Duolink PLA signal from TREK-1 and NHERF-1 was observed in astrocytes compared to only the exposed anti-TREK-1 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChannel activity is associated with surface expression in cells, and this concept commonly serves as a mechanism for regulating channel activity \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. To determine the effect of NHERF-1 on the surface expression of the TREK-1 channel, we preferentially confirmed the surface expression of TREK-1 in NHERF-1 overexpressed or NHERF-1 knocking down cultured astrocytes. Cell surface biotinylation assay indicated that the surface expression of TREK-1 was markedly enhanced in the NHERF-1 knockdown condition using Nherf-1 shRNA in cultured astrocytes (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). We confirm the reverse effect by testing it under the NHERF-1 overexpressed condition. NHERF-1 overexpression significantly reduced the surface expression of TREK-1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and F). These results suggest that NHERF-1 inversely regulates cell surface expression of TREK-1 through physical interactions in cultured astrocytes.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eAstrocytic passive conductance is enhanced in heterozygous NHERF-1 knockout mice\u003c/h2\u003e \u003cp\u003eTo clarify whether NHERF-1 mediates astrocytic passive conductance via TWIK-1/TREK-1 heterodimeric channels in vivo, we used the NHERF-1 gene knockout (KO) animal model. The genomic illustration of NHERF-1 KO mice showed that homologous recombination replaced the first exon containing the beginning of the transcription of the mouse Nherf-1 gene with the neomycin sequence (Figure S5A) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To verify the genetic deletion of NHERF-1 mice, we performed PCR-based genotyping of tail samples from NHERF-1 wild-type (WT) and heterozygous (Het) mice (Figure S5B). qRT-PCR analysis revealed the mRNA expression levels of Nherf-1. In NHERF-1 Het mice, qRT-PCR results confirmed that Nherf-1 mRNA was reduced by almost half (Figures S5C and D). We also examined the expression level of NHERF-1 protein in brain tissue using a specific anti-NHERF-1 antibody (Figure S5E)\u003c/p\u003e \u003cp\u003eTo determine the contribution of NHERF-1 to TWIK-1/TREK-1 heterodimeric channel-mediated astrocytic passive conductance, we aimed to measure the effects of NHERF-1 on astrocytic passive conductance in hippocampal slices of NHERF-1 Het mice. The electrophysiological recording clearly showed that passive conductance in NHERF-1 Het mice was more enhanced than that in WT mice. The application of spadin was significantly modulated in both WT and Het mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The linear current-to-voltage (I-V) relationship of passive conductance reflects the intrinsic properties of mature astrocytes \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. NHERF-1 Het mice also showed an increased linear I-V compared with WT mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C). The spadin-sensitive currents were consistent with these results in the NHERF-1 WT and Het astrocytes (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and E). Together, these results suggest that NHERF-1 regulates TWIK-1/TREK-1 heterodimeric channels-mediated astrocytic passive conductance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eDysfunction of passive conductance by astrocyte-specific NHERF-1 overexpression accelerates seizure susceptibility\u003c/h2\u003e \u003cp\u003eAmong the previous reports, the expression of NHERF-1 is exhibited in various cells of the brain such as ependymal epitheliums \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Because the NHERF-1 KO can't exclude the effects of another cell type, we injected the NHERF-1 overexpressing virus using astrocyte-specific truncated GFAP (GfaABC1D) promoter into the hippocampal CA1 stratum radiatum (SR) regions for 2 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The expression of mCherry protein was observed only in astrocytes, not neurons, of the hippocampal tissue infected with the AAV-GFAPp-NHERF-1-2A-mCherry virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Next, we investigated whether NHERF-1 regulated passive conductance in hippocampal astrocytes. The electrophysiological recording exhibited a huge passive conductance in astrocytes infected AAV-GFAPp-mCherry virus, but when NHERF-1 was overexpressed by AAV-GFAPp-NHERF-1-2A-mCherry virus, passive conductance was dramatically decreased. The reduction of astrocytic passive conductance by NHERF-1 overexpression had no effect after Spadin treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-E). The spadin-sensitive currents were coherent with these results (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF and G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDysregulation of extracellular K\u0026thinsp;+\u0026thinsp;concentration via astrocytic potassium buffering involved in epileptic seizure \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To identify the seizure sensitivity when NHERF-1 overexpressed in astrocytes, which showed decreased astrocytic passive conductance, we observed seizure behavior following the intraperitoneally (i.p) injection of 35 mg/kg kainic acid (KA) in the AAV-GFAPp-mCherry or AAV-GFAPp-NHERF-1-2A-mCherry virus-infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). The behavioral seizure responses were monitored using a modified Racine score for 90 minutes after the KA injection \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. KA-induced seizure responses were bigger in AAV-GFAPp-NHERF-1-2A-mCherry virus-infected mice than in control mice during 90 mins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). We also found that the cumulative score of NHERF-1 overexpressed mice was higher than control mice. But latency to score 3 was not altered in between both mice. (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ and K). These data suggested that When NHERF-1 is overexpressed only in astrocytes, the seizure response is severe because of the reduction of astrocytic passive conductance.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAdditional overexpression of TREK-1 N4 effectively reverses the effects of NHERF-1 overexpression\u003c/h3\u003e\n\u003cp\u003eNext, we asked whether TREK-1 N4, the interaction region of TREK-1 and NHERF-1, prevented the dysfunction of passive conductance and KA-induced seizure susceptibility in NHERF-1 overexpression mice. Before the in vivo experiments, we confirmed the recovery ability by TREK-1 N4 of K\u0026thinsp;+\u0026thinsp;currents under the cultured system. K\u0026thinsp;+\u0026thinsp;currents of mCherry-NHERF-1 transfected astrocytes were decreased, whereas they recovered by HA-TREK-1-2A-GFP overexpression (Figures S6A and B). Next, we generated TREK-1 N4 overexpression virus using GFAP (GfaABC1D) promoter and injected AAV-GFAPp-NHERF-1-2A-mCherry and/or AAV-GFAPp-TREK-1 N4 2A-GFP virus into the hippocampal CA1 stratum radiatum (SR) regions for 2 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The expression mCherry and GFP protein was observed only in astrocytes, not neurons, of the hippocampal tissue infected with the AAV-GFAPp-NHERF-1-2A-mCherry virus and/or AAV-GFAPp-TREK-1 N4-2A-GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, S7A and B). Same as the cultured system, reduced TWIK-1/TREK-1-mediated passive conductance in NHERF-1 astrocyte-specific overexpressed mice was rescued by AAV-GFAPp-TREK-1 N4-2A-GFP virus infection (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-G). The KA-induced behavioral seizure responses during the 90 min observation indicated that AAV-GFAPp-NHERF-1-2A-mCherry and AAV-GFAPp-TREK-1 N4-2A-GFP-infected mice showed delayed seizure onset compared to AAV-GFAPp-NHERF-1-2A-mCherry only infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH and I). The cumulative score for 20 mins also decreased in AAV-GFAPp-NHERF-1-2A-mCherry and AAV-GFAPp-TREK-1 N4-2A-GFP-infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ). However, latency to score 3 was not changed between both mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). These results highlight that the decreased potassium conductance of astrocytes and increased seizure susceptibility caused by NHERF-1 overexpression were restored by additional TREK-1 N4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we found that NHERF-1 is abundantly expressed in astrocytes and not only regulates intrinsic K\u0026thinsp;+\u0026thinsp;currents in cultured cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) but also contributes to TWIK-1/TREK-1-mediated passive conductance in adults (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, we discovered NHERF-1 as a novel binding protein for the TREK-1 channel and confirmed its protein-protein interaction in vitro (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Moreover, NEHRF-1 was associated with endogenous TREK-1 in cultured astrocytes, and overexpression or knockdown of NHERF-1 regulated the surface expression of TREK-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Astrocyte passive conductance was increased in NHERF-1 het KO mice with reduced expression of NHERF-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), whereas NHERF-1 overexpression had the opposite effect. Astrocyte-selective overexpression of NHERF-1 reduced astrocyte passive conductance and predisposed KA-evoked seizure responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, this phenomenon is significantly restored by TREK-1 N4, which interferes with the binding of NHERF-1 to TREK-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The present study demonstrates that NHERF-1 can act as an effective regulator of TREK-1-mediated passive conductance in glial cells. Additionally, it provides evidence that changes in NHERF-1 expression can influence KA-induced seizure sensitivity through alterations in astrocytic passive conductance.\u003c/p\u003e \u003cp\u003eOne of the unique functions of astrocytes is the regulation of ionic homeostasis in the brain, such as extracellular K\u0026thinsp;+\u0026thinsp;buffering through K\u0026thinsp;+\u0026thinsp;channels. Astrocytes show a linear I-V relationship with a negative membrane potential (Vm), called passive conductance. This phenomenon results from leakage of K\u0026thinsp;+\u0026thinsp;membrane conductance, and it is not affected by time and voltage \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Although the exact molecular mechanism is not yet known, it appears to be partly due to weakly rectifying K + (Kir) channels and two-pore domain K + (K2P) channels, especially the TWIK-1/TREK-1 heterodimer channel \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. These changes in ionic homeostasis can induce a variety of symptoms. Mice lacking Kir4.1 in astrocytes show no change in passive conductance but rapidly hyperpolarize the membrane potential of astrocytes and develop severe spontaneous seizures after birth \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, TWIK-1/TREK-1 double KO mice not only did not show the expected changes in astrocytic passive conductance but also showed no pathological behavior \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Since this may be due to a compensatory effect during development, we thought that experiments using viruses in adults would be a more reliable alternative to confirm the role of effective passive conductance. In this study, we used an NHERF-1 overexpressing virus in adults and observed that it can regulate astrocytic passive conductance through interaction with TREK-1. NHERF-1 is a member of the PDZ family, which represents the most common protein-protein interaction domain \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Subcellular localization, endocytosis, transport, and signaling of many receptors and channels are regulated through interaction with PDZ domains in various cells or tissues \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. We demonstrated that TREK-1 binds to the PDZ domain of NHERF-1 and that this interaction regulates spadin-sensitive astrocytic passive conductance (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings suggest the possibility that TREK-1 may interact with other families of proteins that possess PDZ domains in addition to NHERF-1.\u003c/p\u003e \u003cp\u003eTREK-1 has a short N-terminus consisting of 46 amino acids and most of the reported TREK-1 binding proteins bind to its relatively long C-terminus. Interestingly, in this study, NHERF-1 was identified as the protein that binds to the N-terminus of TREK-1, next to β-COP. β-COP was identified as an interacting protein of TREK-1 in astrocytes, binding to the N-terminal domain and increasing both surface expression and channel activity \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, NHERF-1 is bound to the N-terminal domain of TREK-1 but reduces both surface expression and channel activity. Because both of these proteins bind to the TREK-1 N-terminal domain, especially the N4 region, and induce opposite effects, it may be important which protein binds predominantly in a physiological condition. We observed no changes in K\u0026thinsp;+\u0026thinsp;currents when only N4 was expressed in cultured astrocytes (data not shown). If NHERF-1 expression is relatively increased in certain circumstances, such as cancer development, a decrease in TREK-1-mediated K\u0026thinsp;+\u0026thinsp;currents in astrocytes is expected (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). It has been reported that many glioblastoma patients suffer from headaches and seizures \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and one of the reasons for this may be the increased seizure susceptibility caused by NHERF-1 overexpression in astrocytes, according to our results. Further studies are needed to determine the causal relationship between increased expression of NHERF-1 and pathological conditions.\u003c/p\u003e \u003cp\u003eNHERF-1 is widely distributed in the rodent brain and is abundantly expressed in astrocytes rather than neurons. This is crucial because astrocytes are a specific group of glial cells that have functions such as the control of ionic and osmotic composition of the extracellular environment. NHERF-1 in astrocytes supports a role for the PDZ domain as a possible regulator of membrane transporters and channels. In a previous report, excitatory amino acid transporter 1 (EAAT1), called GLAST, colocalized and bound with NHERF-1 in astrocytes. This suggests that NHERF-1 may regulate glutamate homeostasis through GLAST in astrocytes \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Additionally, glial fibrillary acidic protein (GFAP), a characteristic intermediate filament protein of astrocytes, stabilizes and maintains GLAST at the plasma membrane through a series of intermediate linking proteins, including NHERF-1 and ezrin \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Therefore, this report demonstrates a novel function for NHERF-1 in regulating TWIK-1/TREK-1-mediated astrocytic passive conductance. Despite the many roles of NHERF-1 in astrocytes, there are no reports of NHERF-1 being significantly involved in brain function, other than reports that NHERF-1 KO mice exhibit hydrocephalus \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In this respect, this report provides new compelling evidence on the function of NHERF-1 in the brain, especially in astrocytes.\u003c/p\u003e \u003cp\u003eWe also found that the reduced astrocyte passive conductance under NHERF-1 overexpression was restored by TREK-1 N4 addition, and KA-evoked seizure behavior was also significantly rescued (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings suggest that dysfunction of astrocyte passive conductance plays an important role in epileptic seizures and that dissociation of TREK-1 and NHERF-1 complexes should be considered a promising target for seizure therapy. This new target may resolve various side effects caused by existing antiseizure drugs targeting nerve cells. In conclusion, we have identified NHERF-1 as a novel binding protein for TWIK-1/TREK-1 channels and studied the regulatory mechanisms between them, resulting in a better understanding of the function of astrocytes. Additionally, we proposed a new therapeutic strategy targeting astrocytes for the treatment of seizures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation (NRF) of Korea (NRF2022R1A2C1093143), through its funding to J.Y.P. This research was also supported by a grant of NRF of Korea (2020R1A2C2010650) and KIST intramural grant (2E32901), awarded to E.M.H.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e available online.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.Y., J.-Y.P. and E.M.H. designed this study, while J.-Y.P. and E.M.H. supervised and edited the manuscript. Y.B. conducted most of the experiments and acquired the data. A.K. injected AAV and performed behavioral experiments. S. L. conducted most co-IP experiments using overexpressed cells, and S.-S. K. performed co-IP experiments using TREK-1 \u0026Delta;4 mutant overexpressed cells. K.R. conducted co-IP experiments using primary cultured astrocytes. S.P. performed binding analyses using a yeast two hybrid system\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReviewer Information\u003c/strong\u003e included if applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePurushotham, S. S. \u0026amp; Buskila, Y. Astrocytic modulation of neuronal signalling. \u003cem\u003eFrontiers in Network Physiology\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1205544 (2023).\u003c/li\u003e\n\u003cli\u003eKofuji, P. \u0026amp; Newman, E. Potassium buffering in the central nervous system. \u003cem\u003eNeuroscience\u003c/em\u003e \u003cstrong\u003e129\u003c/strong\u003e, 1043-1054 (2004).\u003c/li\u003e\n\u003cli\u003eBellot-Saez, A., K\u0026eacute;kesi, O., Morley, J. W. \u0026amp; Buskila, Y. Astrocytic modulation of neuronal excitability through K+ spatial buffering. \u003cem\u003eNeuroscience \u0026amp; Biobehavioral Reviews\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 87-97 (2017).\u003c/li\u003e\n\u003cli\u003eRyoo, K. \u0026amp; Park, J.-Y. Two-pore domain potassium channels in astrocytes. \u003cem\u003eExperimental neurobiology\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 222 (2016).\u003c/li\u003e\n\u003cli\u003eWang, F.\u003cem\u003e et al.\u003c/em\u003e Astrocytes modulate neural network activity by Ca2+-dependent uptake of extracellular K+. \u003cem\u003eScience signaling\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, ra26-ra26 (2012).\u003c/li\u003e\n\u003cli\u003eZhou, M.\u003cem\u003e et al.\u003c/em\u003e TWIK-1 and TREK-1 are potassium channels contributing significantly to astrocyte passive conductance in rat hippocampal slices. \u003cem\u003eJournal of Neuroscience\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 8551-8564 (2009).\u003c/li\u003e\n\u003cli\u003eMi Hwang, E.\u003cem\u003e et al.\u003c/em\u003e A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes. \u003cem\u003eNature communications\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 3227 (2014).\u003c/li\u003e\n\u003cli\u003eBae, Y.\u003cem\u003e et al.\u003c/em\u003e Spadin modulates astrocytic passive conductance via inhibition of TWIK-1/TREK-1 heterodimeric channels. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 9639 (2020).\u003c/li\u003e\n\u003cli\u003eBushau-Sprinkle, A. M. \u0026amp; Lederer, E. D. New roles of the Na+/H+ exchange regulatory factor 1 scaffolding protein: a review. \u003cem\u003eAmerican Journal of Physiology-Renal Physiology\u003c/em\u003e \u003cstrong\u003e318\u003c/strong\u003e, F804-F808 (2020).\u003c/li\u003e\n\u003cli\u003eDunn, H. A. \u0026amp; Ferguson, S. S. PDZ protein regulation of G protein\u0026ndash;coupled receptor trafficking and signaling pathways. \u003cem\u003eMolecular pharmacology\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 624-639 (2015).\u003c/li\u003e\n\u003cli\u003eVaquero, J., Nguyen Ho-Bouldoires, T., Claperon, A. \u0026amp; Fouassier, L. Role of the PDZ-scaffold protein NHERF1/EBP50 in cancer biology: from signaling regulation to clinical relevance. \u003cem\u003eOncogene\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 3067-3079 (2017).\u003c/li\u003e\n\u003cli\u003eTreat, A. C.\u003cem\u003e et al.\u003c/em\u003e The PDZ protein Na+/H+ exchanger regulatory factor-1 (NHERF1) regulates planar cell polarity and motile cilia organization. \u003cem\u003ePloS one\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e0153144 (2016).\u003c/li\u003e\n\u003cli\u003eEdiger, T. R., Kraus, W. L., Weinman, E. J. \u0026amp; Katzenellenbogen, B. S. Estrogen receptor regulation of the Na+/H+ exchanger regulatory factor. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 2976-2982 (1999).\u003c/li\u003e\n\u003cli\u003eWeinman, E. J., Steplock, D., Wang, Y. \u0026amp; Shenolikar, S. Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na (+)-H+ exchanger. \u003cem\u003eThe Journal of clinical investigation\u003c/em\u003e \u003cstrong\u003e95\u003c/strong\u003e, 2143-2149 (1995).\u003c/li\u003e\n\u003cli\u003eSong, X.\u003cem\u003e et al.\u003c/em\u003e Canonical transient receptor potential channel 4 (TRPC4) co‐localizes with the scaffolding protein ZO‐1 in human fetal astrocytes in culture. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 418-429 (2005).\u003c/li\u003e\n\u003cli\u003eLee, A.\u003cem\u003e et al.\u003c/em\u003e Na+\u0026ndash;H+ exchanger regulatory factor 1 is a PDZ scaffold for the astroglial glutamate transporter GLAST. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 119-129 (2007).\u003c/li\u003e\n\u003cli\u003eRitter-Makinson, S. L.\u003cem\u003e et al.\u003c/em\u003e Group II metabotropic glutamate receptor interactions with NHERF scaffold proteins: Implications for receptor localization in brain. \u003cem\u003eNeuroscience\u003c/em\u003e \u003cstrong\u003e353\u003c/strong\u003e, 58-75 (2017).\u003c/li\u003e\n\u003cli\u003eBhattacharya, S., Stanley, C. B., Heller, W. T., Friedman, P. A. \u0026amp; Bu, Z. Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e294\u003c/strong\u003e, 11297-11310 (2019).\u003c/li\u003e\n\u003cli\u003eShibata, T., Chuma, M., Kokubu, A., Sakamoto, M. \u0026amp; Hirohashi, S. EBP50, a \u0026beta;-catenin-associating protein, enhances Wnt signaling and is over-expressed in hepatocellular carcinoma. \u003cem\u003eHepatology\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 178-186 (2003).\u003c/li\u003e\n\u003cli\u003eKislin, K. L., McDonough, W. S., Eschbacher, J. M., Armstrong, B. A. \u0026amp; Berens, M. E. NHERF-1: modulator of glioblastoma cell migration and invasion. \u003cem\u003eNeoplasia\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 377-IN377 (2009).\u003c/li\u003e\n\u003cli\u003eRacine, R. J. Modification of seizure activity by electrical stimulation: cortical areas. \u003cem\u003eElectroencephalography and clinical neurophysiology\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 1-12 (1975).\u003c/li\u003e\n\u003cli\u003eStorch, U.\u003cem\u003e et al.\u003c/em\u003e Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, E37-E46 (2017).\u003c/li\u003e\n\u003cli\u003eMartin, E. R., Barbieri, A., Ford, R. C. \u0026amp; Robinson, R. C. In vivo crystals reveal critical features of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and the PDZ2 domain of Na+/H+ exchange cofactor NHERF1. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e295\u003c/strong\u003e, 4464-4476 (2020).\u003c/li\u003e\n\u003cli\u003eSaha, T.\u003cem\u003e et al.\u003c/em\u003e Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner. \u003cem\u003eBiochemistry and Biophysics Reports\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 100912 (2021).\u003c/li\u003e\n\u003cli\u003eChoe, S. Potassium channel structures. \u003cem\u003eNature Reviews Neuroscience\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 115-121 (2002).\u003c/li\u003e\n\u003cli\u003eEnyedi, P. \u0026amp; Czirj\u0026aacute;k, G. Molecular background of leak K+ currents: two-pore domain potassium channels. \u003cem\u003ePhysiological reviews\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 559-605 (2010).\u003c/li\u003e\n\u003cli\u003eCoetzee, W. A.\u003cem\u003e et al.\u003c/em\u003e Molecular diversity of K+ channels. \u003cem\u003eAnnals of the New York Academy of Sciences\u003c/em\u003e \u003cstrong\u003e868\u003c/strong\u003e, 233-255 (1999).\u003c/li\u003e\n\u003cli\u003eLesage, F.\u003cem\u003e et al.\u003c/em\u003e TWIK‐1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure. \u003cem\u003eThe EMBO journal\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1004-1011 (1996).\u003c/li\u003e\n\u003cli\u003eKim, S.-S.\u003cem\u003e et al.\u003c/em\u003e \u0026beta;-COP Regulates TWIK1/TREK1 Heterodimeric Channel-Mediated Passive Conductance in Astrocytes. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 3322 (2022).\u003c/li\u003e\n\u003cli\u003eSol\u0026eacute;, L.\u003cem\u003e et al.\u003c/em\u003e KCNE4 suppresses Kv1. 3 currents by modulating trafficking, surface expression and channel gating. \u003cem\u003eJournal of cell science\u003c/em\u003e \u003cstrong\u003e122\u003c/strong\u003e, 3738-3748 (2009).\u003c/li\u003e\n\u003cli\u003eLee, Y.-S.\u003cem\u003e et al.\u003c/em\u003e Suppression of 14-3-3\u0026gamma;-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells. \u003cem\u003eScientific reports\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 26413 (2016).\u003c/li\u003e\n\u003cli\u003eShenolikar, S., Voltz, J., Minkoff, C., Wade, J. \u0026amp; Weinman, E. Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 11470-11475 (2002).\u003c/li\u003e\n\u003cli\u003eWang, F., Qi, X., Zhang, J. \u0026amp; Huang, J. H. Astrocytic modulation of potassium under seizures. \u003cem\u003eNeural Regeneration Research\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 980 (2020).\u003c/li\u003e\n\u003cli\u003eDavid, Y.\u003cem\u003e et al.\u003c/em\u003e Astrocytic dysfunction in epileptogenesis: consequence of altered potassium and glutamate homeostasis? \u003cem\u003eJournal of Neuroscience\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 10588-10599 (2009).\u003c/li\u003e\n\u003cli\u003eNwaobi, S. E., Cuddapah, V. A., Patterson, K. C., Randolph, A. C. \u0026amp; Olsen, M. L. The role of glial-specific Kir4. 1 in normal and pathological states of the CNS. \u003cem\u003eActa neuropathologica\u003c/em\u003e \u003cstrong\u003e132\u003c/strong\u003e, 1-21 (2016).\u003c/li\u003e\n\u003cli\u003eOlsen, M. L.\u003cem\u003e et al.\u003c/em\u003e New insights on astrocyte ion channels: critical for homeostasis and neuron-glia signaling. \u003cem\u003eJournal of Neuroscience\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 13827-13835 (2015).\u003c/li\u003e\n\u003cli\u003eDjukic, B., Casper, K. B., Philpot, B. D., Chin, L.-S. \u0026amp; McCarthy, K. D. Conditional knock-out of Kir4. 1 leads to glial membrane depolarization, inhibition of potassium and glutamate uptake, and enhanced short-term synaptic potentiation. \u003cem\u003eJournal of Neuroscience\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 11354-11365 (2007).\u003c/li\u003e\n\u003cli\u003eDu, Y.\u003cem\u003e et al.\u003c/em\u003e Genetic deletion of TREK-1 or TWIK-1/TREK-1 potassium channels does not alter the basic electrophysiological properties of mature hippocampal astrocytes in situ. \u003cem\u003eFrontiers in cellular neuroscience\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 13 (2016).\u003c/li\u003e\n\u003cli\u003eFeng, W. \u0026amp; Zhang, M. Organization and dynamics of PDZ-domain-related supramodules in the postsynaptic density. \u003cem\u003eNature Reviews Neuroscience\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 87-99 (2009).\u003c/li\u003e\n\u003cli\u003eLee, H.-J. \u0026amp; Zheng, J. J. PDZ domains and their binding partners: structure, specificity, and modification. \u003cem\u003eCell communication and Signaling\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 1-18 (2010).\u003c/li\u003e\n\u003cli\u003eKim, E.\u003cem\u003e et al.\u003c/em\u003e Enhancement of TREK1 channel surface expression by protein\u0026ndash;protein interaction with \u0026beta;-COP. \u003cem\u003eBiochemical and biophysical research communications\u003c/em\u003e \u003cstrong\u003e395\u003c/strong\u003e, 244-250 (2010).\u003c/li\u003e\n\u003cli\u003ePace, A.\u003cem\u003e et al.\u003c/em\u003e End of life issues in brain tumor patients. \u003cem\u003eJournal of neuro-oncology\u003c/em\u003e \u003cstrong\u003e91\u003c/strong\u003e, 39-43 (2009).\u003c/li\u003e\n\u003cli\u003eSizoo, E. M.\u003cem\u003e et al.\u003c/em\u003e Symptoms and problems in the end-of-life phase of high-grade glioma patients. \u003cem\u003eNeuro-oncology\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1162-1166 (2010).\u003c/li\u003e\n\u003cli\u003eSullivan, S. M.\u003cem\u003e et al.\u003c/em\u003e Cytoskeletal anchoring of GLAST determines susceptibility to brain damage: an identified role for GFAP. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e282\u003c/strong\u003e, 29414-29423 (2007).\u003c/li\u003e\n\u003cli\u003eGeorgescu, M.-M.\u003cem\u003e et al.\u003c/em\u003e NHERF1/EBP50 is an organizer of polarity structures and a diagnostic marker in ependymoma. \u003cem\u003eActa neuropathologica communications\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1-10 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"astrocytic passive conductance, TREK-1, NHERF-1, kainic acid-induced seizures","lastPublishedDoi":"10.21203/rs.3.rs-3974699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3974699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMature hippocampal astrocytes exhibit a linear current-to-voltage (I-V) K\u0026thinsp;+\u0026thinsp;membrane conductance, which is called passive conductance. It is estimated to enable astrocytes to keep potassium homeostasis in the brain. We previously reported that the TWIK-1/TREK-1 heterodimeric channels are crucial for astrocytic passive conductance. However, the regulatory mechanism of these channels by other binding proteins still remains elusive. Here, we identified Na+/H\u0026thinsp;+\u0026thinsp;exchange regulator-1 (NHERF-1), a protein highly expressed in astrocytes, as a candidate interaction partner for these channels. NHERF-1 endogenously bound to TWIK-1/TREK-1 in hippocampal cultured astrocytes. When NHERF-1 is overexpressed or silenced, surface expression and activity of TWIK-1/TREK-1 heterodimeric channels were inhibited or enhanced, respectively. Furthermore, we confirmed that reduced astrocytic passive conductance by NHERF-1 overexpressing in the hippocampus increases kainic acid (KA)-induced seizure sensitivity. Taken together, these results suggest that NHERF-1 is a key regulator of TWIK-1/TREK-1 heterodimeric channels in astrocytes and suppression of TREK-1 surface expression by NHERF-1 increases KA-induced seizure susceptibility via reduction of astrocytic passive conductance.\u003c/p\u003e","manuscriptTitle":"Astrocytic NHERF-1 increases seizure susceptibility by inhibiting surface expression of TREK-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-06 10:32:10","doi":"10.21203/rs.3.rs-3974699/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c5c12945-b441-4653-826e-478d7c23f2c4","owner":[],"postedDate":"March 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-27T05:06:05+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-06 10:32:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3974699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3974699","identity":"rs-3974699","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}