Naunyn-Schmiedeberg's Arch Pharmacol DOI 10.1007/s00210-013-0952-2

ORIGINAL ARTICLE

Electrophysiology and pharmacology of tandem domain potassium channel TREK-1 related BDNF synthesis in rat astrocytes Li Lu & Weiping Wang & Ying Peng & Jiang Li & Ling Wang & Xiaoliang Wang

Received: 11 October 2013 / Accepted: 18 December 2013 # Springer-Verlag Berlin Heidelberg 2014

Abstract In the present study, the functional properties and pharmacology of two-pore domain potassium channel (K2P) TREK-1 in primary cultured rat brain astrocytes were investigated. Western blot, patch clamping techniques, and ELISA were used to detect the distribution and function of TREK-1 as well as the expression of brain-derived neurotrophic factor (BDNF) on the primary cultured astrocytes. It was shown that TREK-1 protein expressed in astrocytes was 2.4-fold higher than it was expressed in microglia. Single channel recording via patch clamping showed that the TREK-1 outward currents in astrocytes could be activated by arachidonic acid (AA) or chloroform with the conductance of 113±14 and 120±13 pS, respectively. The current was also sensitive to mechanical stretch and intracellular acidification. Negative pressure (−30 cm H2O) and acidification of intracellular solution (pH 6.8 or 6.3) both enhanced TREK-1 channel open probability significantly. Further pharmacological studies showed that TREK-1 antagonist penfluridol inhibited AA-induced currents, and both penfluridol and methionine (TREK-1 blockers) significantly increased BDNF level in astrocytes by 50 %. These results indicated that TREK-1 channel current was a major component of K2P currents in astrocytes. TREK-1 channels might play important roles in regulating the function of astrocytes and might be used as a drug target for neuroprotection. Keywords TREK-1 . Astrocyte . Potassium channels . BDNF . Single channel recording L. Lu : W. Wang : Y. Peng : J. Li : L. Wang : X. Wang (*) State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Xiannongtan Street, Xicheng District, Beijing 100050, China e-mail: [email protected] L. Lu Current address: Pharmacology Research Institute of Basic Medical School, Lanzhou University, Lanzhou 730000, China

Abbreviations AA BDNF K2P CNS GFAP

Arachidonic acid Brain-derived neurotrophic factor Tandem domain potassium channel Central nervous system Glial fibrillary acidic protein

Introduction Astrocytes, the most abundant glial cell type, are ubiquitous throughout all regions of the central nervous system (CNS). For decades, astrocytes have been regarded as passive partners of neurons in the functional performance of the CNS. However, this viewpoint was challenged by increasing evidences that astrocytes play an primary role in neural processing (Fuller et al. 2010). Astrocytes provide the metabolic and trophic support to neurons, participate in the synaptic function and plasticity, and maintain the extracellular balance of ions, fluid, and transmitters (Rossi and Volterra 2009). In addition, astrocytes respond to a variety of injury of the CNS, such as cerebral ischemia, neurodegenerative disease, and infection. Under physiological conditions, astrocytes show a more hyperpolarized membrane potential than neurons and provide a driving force for their function. It has been reported that highly hyperpolarized membrane potential of astrocytes is largely due to the presence of Kir4.1 inward rectifying K+ channels (Kucheryavykh et al. 2009; Seifert et al. 2009). However, Kir4.1 is not the only channel type contributing to the overall negative membrane potential of astrocytes, because inward currents were dramatically reduced after knockdown of Kir4.1 channels, whereas robust outward currents remained (Kucheryavykh et al. 2009). There must be other background K+ channels functionally expressed in astrocytes.

Naunyn-Schmiedeberg's Arch Pharmacol

Our previous studies showed that TREK-1 was highly expressed in rat brain and it was upregulated during acute and chronic brain ischemia (Xu et al. 2004; Li et al. 2005). It indicated an important role of TREK-1 in neuronal injury and neuroprotection. Meanwhile, TREK-1 channels were reported to express widely in the CNS, including the cortex, hippocampus, cerebellum, and hypothalamus (Meadows et al. 2000; Talley et al. 2001). Recently, several reports manifested the expression and partial function of TREK-1 in astrocytes (Seifert et al. 2009; Zhou et al. 2009; Wu et al. 2013). TREK-1 might be one of the major components of the K+ channels underlying the passive conductance of mature hippocampal astrocytes (Zhou et al. 2009). The TREK-1 activity was also reported to be involved in the function of astrocytes and neuronal survival (Wu et al. 2013). Till now, TREK-1 was the most extensively studied twopore domain potassium channels (K2P), and it is known to mediate the background potassium current which plays an important role in regulating the resting membrane potential and cell excitability. TREK-1 was also known to display low basal activity and can be activated by a number of factors including physical stimulation (stretch, cell swelling, and heat) and chemical stimulation (polyunsaturated fatty acid, intracellular acidosis). In addition, it can be activated by some volatile anesthetic agents and gas molecules (Lesage and Lazdunski 2000; Mathie 2007). Recently, studies based on genetic knockout mice have shown that TREK-1 potassium channel may participate in neuroprotection, epilepsy, pain perception, and depression processes (Alloui et al. 2006; Heurteaux et al. 2004; Heurteaux et al. 2006). Due to the important physiological and pathophysiological roles of glias, TREK-1 as a modulator of glia remained to be studied. However, the relative importance and the differential expression of TREK-1 in astrocytes and microglias were not compared at the same time. The characterization of TREK-1 single channel recording on astrocytes was not reported yet. In addition, the relationship between the electrophysiological function and the pharmacology of TREK-1, especially TREK-1 as a drug target-mediated BDNF synthesis in astrocytes, remained to be studied so far. Thus, the aims of the present study were to elucidate the different expressions of TREK-1 in gliacytes and to reveal the electrophysiological properties of TREK-1 regulated by its activators and blockers as well as to demonstrate TREK-1 blocker-induced BDNF synthesis in astrocytes.

Materials and methods Primary culture of glial cells Glial cells were cultured and purified as described previously (Jana et al. 2007). Pups of neonatal 24-h Wistar rats were

decapitated and the cerebral hemispheres were immediately transferred to cold DMEM/F12 media, and then the meninges were carefully removed under a dissecting microscope. The cerebral tissue was treated with a 0.25 % trypsin solution for 30 min at 37 °C. An equal volume of DMEM/F12 medium containing 10 % FBS was added to stop the trypsin, and the mixture was centrifuged at 800×g for 3–5 min. The pellet was resuspended and passed through a stainless steel mesh (75 μm pore size, 200-mesh screen). The cultures were incubated at 37 °C in a humid 5 % CO2/95 % air environment. The complete medium was changed after 24 h and half of the medium was changed every 3 days. Purification of astrocytes and microglia After 2 weeks, mixed glial cultures were subjected to shaking to take advantage of the selective cell adhesion properties of the different glial subtypes as previously described (Giulian 1986). The cultures were placed on a rotary shaker at 180 rpm at 37 °C for 5 h to remove loosely attached microglia. The cell suspensions were centrifuged, resuspended, and replanted in 10 cm dishes. The attached cells remaining after the removal of microglia were primarily the astrocytes. These cells were trypsinized and subcultured twice in complete medium to yield more viable and healthy cells in a week. The purified astrocytes and microglia were stained with the antibody against glial fibrillary acidic protein (GFAP) or CD11b (Marek et al. 2008). More than 90 % of these cells stained for the microglial marker CD11b. More than 95 % of these cells obtained by this method were found to be positive for GFAP, a marker for astrocytes. Western blot To determine the TREK-1 protein expression, whole-cell protein extracts were prepared from astrocytes and microglia. Cells were washed twice with PBS and solubilized in a lysis buffer (150 mM NaCl, 10 mM Tris–HCl (pH 7.0), 1 mM EGTA, 1 % (v/v) NP40, 10 % (v/v) glycerol, 1 mg/ml cocktail). Protein concentrations were determined by the Coomassie blue protein assay. Protein samples (50 μg) were separated by 10 % SDS/PAGE and electrotransferred on to a nitrocellulose membrane. After blocking at room temperature in 5 % milk with TBST buffer (10 mM Tris–HCl, 120 mM NaCl, 0.1 % Tween-20, pH 7.4) for 1 h, the membrane was incubated with TREK-1 antibody (1:200, Sigma, Saint Louis, MO, USA) at 4 °C overnight. Membranes were washed three times in TBST buffer, followed by incubation with 1:10,000 dilutions of HRP-conjugated anti-rabbit IgG at room temperature for 1 h, and washed three times in TBST. Proteins were visualized by using an advanced Chemiluminescence® (ECL) kit. The density of the bands on Western blots was

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quantified by densitometry analysis of the scanned blots using ImageQuant software (Bio-Rad, Hercules, CA, USA). Electrophysiological studies For cell-attached and inside-out recording, both pipette and bath solution contained 140 mM KCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (pH 7.3). Patch pipettes were heat-polished to produce tip diameters of about 1 μm with about 10 MΩ resistance when filled with pipette solutions. To examine the effects of mechanical stress on the activity of TREK-like channels, we elicited membrane stretch (negative pressure) by applying a suction through a syringe. The pressure was monitored with a manometer. In order to indentify a characteristic of TREK-like potassium channel in the native astrocytes, 10 mM TEA, 1 mM BaCl 2 , and 10 μm glibenclamide were added into the bath solution to avoid the effects of other potassium channels. Preparation and storage of AA and chloroform was according to a previous research (Terrenoire et al. 2001). Currents were obtained with an EPC-10 patch-clamp amplifier (Heka Electronic, Lambrecht, Pfalz, Germany), filtered at 2 kHz, and digitized at 10 kHz. Single channel recordings were analyzed using TAC+TACFit (X4.0.9, Bruxton, Seattle, WA, USA). Openings of the channel were detected using half-amplitude threshold analysis. Open probability was calculated from the total open times divided by a total of 2 s sweep duration in the normal control group (control groups in Figs. 3 and 5) and divided by a total 3 s sweep duration in the treated groups (Figs. 3 and 5). The total recording time was more than 10 s and 2 or 3 s was used for calculation of the Po. Four cells (patches) were recorded for each group. All experiments were carried out at room temperature (22–24 °C). Extraction of BDNF To determine the expression levels of brain-derived neurotrophic factor (BDNF) in response to TREK-1 inhibitors, astrocytes were seeded at a density of 5×105 cells/ml in 48well culture plates (Corning Incorporated, NY) and grown for 24 h. Then the cells were treated with TREK-1 inhibitors, penfluridol or methionine (Sigma, Saint Louis, MO, USA) for 12 h. Following the desired incubation period, the cells were harvested in 5 ml of ice-cold PBS and centrifuged at 4 °C for 5 min at 3,000×g. Cell samples were prepared by sonication of pellets for 10 s at approximately 40 W in a lysis buffer. The samples were then centrifuged at 12,000×g for 10 min at 4 °C. The supernatants were frozen at −20 °C until used. BDNF enzyme immunoassay BDNF levels were measured by ELISA (Rat BDNF ELISA Kit, Boster, China) according to the manufacturer’s

instructions. Briefly, 50 μl of the sample or standard (0– 2,000 pg BDNF/ml) was added to the wells which the capture antibody was precoated in duplicate and incubated for 90 min at 37 °C, followed by washing with the appropriate buffer. Plates were incubated with anti-rat BDNF monoclonal antibody at 37 °C for 2 h, washed, and then incubated with streptavidin-HRP for 1 h. Finally, the plates were incubated in tetramethylbenzidine solution to produce a color reaction. The reaction was stopped with 1 M H 2 SO 4 . The absorbance of the reaction product was measured by a microplate reader in the time span of less than 15 min at 450 nm. The concentration of BDNF in the samples was calculated from the BDNF standard curve performed on each microtiter plate subtracting the nonspecific signal from all obtained values. Statistical analysis All values were expressed as means±SEM. Statistical significance was evaluated by Student’s t test or one-way analysis of variance (ANOVA) using SPSS10.0 (SPSS Inc. Chicago, IL, USA). p