• Journal of the Korea Academia-Industrial cooperation Society
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• v.13 no.5
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• pp.2125-2132
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• 2012

Fluoxetine and tianeptine are commonly used as antidepressants (AD), and haloperidol and risperidone are widely used as antipsychotic drugs (APD), and it modulates various ion channels. TREK2 channel subfamily is very similar to physiological properties of TREK1 channel which can play important roles in the pathophysiology of mental disorders such as depression and schizophrenia, therefore, the pharmacological effect of psychiatric and depression drug on TREK2 channel may be similar to those of TREK1. Using the excised inside-out patch-clamp technique, we have examined the effects of APD and AD on cloned TREK2 channel expressed CHO cells. Fluoxetine (selective serotonin release inhibitor, SSRI) inhibited the TREK2 channel in a concentration-dependent manner ($IC_{50}$ $13{\mu}M$), whereas selective serotonin reuptake enhancer (SSRE) tianeptine increased without reducing the TREK2 channel activity. Haloperidol also inhibited the TREK2 channel in a concentration-dependent manner ($IC_{50}$ $44{\mu}M$), whereas even higher concentration ($100{\mu}M$) of risperidone did not completely inhibit on the activity. This study showed that TREK2 channel was preferentially blocked by fluoxetine rather than tianeptine, and inhibited by haloperidol rather than risperidone, suggesting differential effect of TREK2 channels by APD and AD may contribute to some mechanism of adverse side effects.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.12 no.6
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• pp.2660-2667
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• 2011

TREK-1 channel is a member of the two-pore domain potassium (K2P) channel family that is regulated by intracellular pH, membrane stretch, polyunsaturated fatty acids, temperature, and some neuroprotectant agents. TREK-1 channel can influence neuronal excitability by regulating leakage of potassium ions and resting membrane potential. TREK-1 channel has been shown to be overexpressed in prostate cancer cells. Although the importance of these properties, relatively little is known about flavonoid effects in the regulations of TREK-1 channel. The purpose of the study was to screening of flavonoids as the TREK-1 channel modulator using one of electrophysiological techniques such as excised inside-out patch configuration. We demonstrated blocking effect on TREK-1 channel by flavonoids such as epigallocatechin-3-gallate (EGCG), curcumin and quercetin in CHO cells transiently expressing TREK-1 channel. The inhibition of TREK-1 channel by quercetin and curcumin was reversible, whereas EGCG was little reversible. Quercetin, EGCG and curcumin decreased the relative channel activity to 73%, 91% and 94%, respectively. The half-inhibitory concentration (IC50) of curcumin, quercetin and EGCG was $1.04{\pm}0.19\;{\mu}M$, $1.13{\pm}0.26\;{\mu}M$ and $13.5{\pm}2.20\;{\mu}M$ in CHO cells expressing TREK-1 channel, respectively. These results indicate that flavonoids might regulate TREK-1 and this regulation might be one of the pharmacological actions of flavonoid in nervous systems and cancer cells.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.4
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• pp.211-216
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• 2008

TREK (TWIK-RElated $K^+$ channels) and TRAAK (TWIK-Related Arachidonic acid Activated $K^+$ channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of - 40 mV in symmetrical 150 mM $K^+$ solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.

• Biomolecules & Therapeutics
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• v.24 no.3
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• pp.252-259
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• 2016

Neuropathic pain is a complex state showing increased pain response with dysfunctional inhibitory neurotransmission. The TREK family, one of the two pore domain $K^+$ (K2P) channel subgroups were focused among various mechanisms of neuropathic pain. These channels influence neuronal excitability and are thought to be related in mechano/thermosensation. However, only a little is known about the expression and role of TREK-1 and TREK-2, in neuropathic pain. It is performed to know whether TREK-1 and/or 2 are positively related in dorsal root ganglion (DRG) of a mouse neuropathic pain model, the chronic constriction injury (CCI) model. Following this purpose, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and western blot analyses were performed using mouse DRG of CCI model and compared to the sham surgery group. Immunofluorescence staining of isolectin-B4 (IB4) and TREK were performed. Electrophysiological recordings of single channel currents were analyzed to obtain the information about the channel. Interactions with known TREK activators were tested to confirm the expression. While both TREK-1 and TREK-2 mRNA were significantly overexpressed in DRG of CCI mice, only TREK-1 showed significant increase (~9 fold) in western blot analysis. The TREK-1-like channel recorded in DRG neurons of the CCI mouse showed similar current-voltage relationship and conductance to TREK-1. It was easily activated by low pH solution (pH 6.3), negative pressure, and riluzole. Immunofluorescence images showed the expression of TREK-1 was stronger compared to TREK-2 on IB4 positive neurons. These results suggest that modulation of the TREK-1 channel may have beneficial analgesic effects in neuropathic pain patients.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.4
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• pp.379-385
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• 2016

TWIK-related $K^+$ channel-2 (TREK-2) and TWIK-related spinal cord $K^+$ (TRESK) channel are members of two-pore domain $K^+$ channel family. They are well expressed and help to set the resting membrane potential in sensory neurons. Modulation of TREK-2 and TRESK channels are involved in the pathogenesis of pain, and specific activators of TREK-2 and TRESK may be beneficial for the treatment of pain symptoms. However, the effect of commonly used analgesics on TREK-2 and TRESK channels are not known. Here, we investigated the effect of analgesics on TREK-2 and TRESK channels. The effects of analgesics were examined in HEK cells transfected with TREK-2 or TRESK. Amitriptyline, citalopram, escitalopram, and fluoxetine significantly inhibited TREK-2 and TRESK currents in HEK cells (p<0.05, n=10). Acetaminophen, ibuprofen, nabumetone, and bupropion inhibited TRESK, but had no effect on TREK-2. These results show that all analgesics tested in this study inhibit TRESK activity. Further study is needed to identify the mechanisms by which the analgesics modulate TREK-2 and TRESK differently.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.17 no.3
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• pp.127-135
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• 2016

Two-pore domain potassium (K2P) channels are the targets of physiological stimuli, such as intracellular pH, bioactive lipids, and neurotransmitters, and they set the resting membrane potential. Some types of K2P channels play a critical role in both apoptosis and tumoriogenesis. Among the K2P channels, no antagonists of the TREK2 channel have been reported. The aim of the present study was to determine if the TREK2 channel is blocked and whether cell proliferation is influenced by flavonoids in the TREK2 overexpressing HEK293 cells (HEKT2). The electrophysiological current was recorded using single channel patch clamp techniques and cell proliferation was measured using a XTT assay. The electrophysiological results showed that the TREK2 channel activity was reduced to $91.5{\pm}13.1%$ (n=5) and $82.2{\pm}13.7%$ (n=5) by flavonoids, such as epigallocatechin-3-gallate (EGCG) and quercetin in HEKT2 cells, respectively. In contrast, the EGCG analogue, epicatechin (EC), had no significant inhibitory effects on the TREK2 single channel activity. In addition, cell proliferation was reduced to $69.4{\pm}14.0%$ (n=4) by ECGG in the HEKT2 cells. From these results, EGCG and quercetin represent the first known TREK2 channel inhibitors and only EGCG reduced HEKT2 cell proliferation. This suggests that the flavonoids may work primarily by inhibiting the TREK2 channel, leading to a change in the resting membrane potential, and triggering the initiation of a change in intracellular signaling for cell proliferation. TREK2 channel may, at least in part, contribute to cell proliferation.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.209-214
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• 2011

Endometrium undergoing hormonal change plays important roles in preparation for implantation, fetal growth, and well-being. During pregnancy, cellular remodeling and hormonal changes in endometrium could change two-pore domain K$^+$ channel (K$_{2P}$) expression. This study was performed to identify whether K$_{2P}$ channel expression is changed in endometrium of pregnant Korean cattle, and whether the expression level is modulated by progesterone treatment. We investigated changes in the mRNA and protein expressions of K$_{2P}$ channel in pregnant endometrium using RT-PCR and Western blot analyses. The expression levels of all K$_{2P}$ channel mRNAs tested in this study, except that of TREK-1, were changed in the pregnant endometrium. mRNA levels of TASK-3 and TRAAK were significantly down-regulated, whereas those of TREK-2 and TRESK were up-regulated in the pregnant endometrium. In parallel with the RT-PCR results, Western blot analysis revealed up-regulations of TREK-2 (7.9-fold) and TRESK (2-fold) proteins levels in the pregnant endometrium. In addition, TREK-2 and TRESK protein levels were up-regulated in bovine endometrial cells by progesterone treatment (10 ${\mu}g$/ml). From these results, we suggest that the up-regulation of TREK-2 and TRESK by progesterone may contribute to the regulation of physiological changes during pregnancy.

• Journal of Embryo Transfer
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• v.22 no.3
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• pp.149-154
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• 2007

Endometrial cells play important roles in implantation and during pregnancy. This study was carried out to identify whether two-pore domain $K^+\;(K_{2P})$ channels are expressed in endometrial cells of Korean cattle. $K_{2P}$ channels set the resting membrane potential of many kinds of neuronal cells in the central and peripheral nervous systems. RT-PCR data showed that TASK-1, TASK-3, TREK-1, TREK-2, and TRAAK were expressed in bovine endometrial cells, and the mRNA expression levels were similar between endometrial cells with or without endometritis. The protein expression was confirmed by using commercially available polyclonal antibodies (TASK-3, TREK-1, TREK-2, and TRAAK). TASK-3 and TREK-1 were expressed in all area of endometrial cells including nuclei, while TREK-2 and TRAAK were expressed in all area of cells except nuclei. These results demonstrate for the first time the presence of $K_{2P}$ channel in endometrial cells of Korean cattle.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.16 no.3
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• pp.1964-1971
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• 2015

Human bone marrow or human umbilical cord vein derived-mesenchymal stem cells (hBM-MSCs or hUC-MSCs) have known as a potentially useful cell type for clinical therapeutic applications. We investigated two-pore domain potassium (K2P) channels in these cells. K2P channels play a major role in setting the resting membrane potential in many cell types. Among them, TREK1 is targets of hydrogen, hypoxia, polyunsaturated fatty acids, antidepressant, and neurotransmitters. We investigated whether hBM-MSCs and hUC-MSCs express functional TREK1 channel using RT-PCR analysis and patch clamp technique. Potassium channel with a single channel conductance of 100 pS was found in hUC-MSCs and BM-MSCs and the channel was activated by membrane stretch (-5 mmHg ~ -15 mmHg), arachidonic acid ($10{\mu}M$) and intracellular acidosis (pH 6.0). These electrophysiological properties were similar to those of TREK1. Our results suggest that TREK1 is functionally present in hBM-MSCs and hUC-MSCs, where they contribute to its resting membrane potential.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.6
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• pp.511-516
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• 2013

Bladder cancer is the seventh most common cancer in men that smoke, and the incidence of disease increases with age. The mechanism of occurrence has not yet been established. Potassium channels have been linked with cell proliferation. Some two-pore domain $K^+$ channels (K2P), such as TASK3 and TREK1, have recently been shown to be overexpressed in cancer cells. Here we focused on the relationship between cell growth and the mechanosensitive K2P channel, TREK2, in the human bladder cancer cell line, 253J. We confirmed that TREK2 was expressed in bladder cancer cell lines by Western blot and quantitative real-time PCR. Using the patch-clamp technique, the mechanosensitive TREK2 channel was recorded in the presence of symmetrical 150 mM KCl solutions. In 253J cells, the TREK2 channel was activated by polyunsaturated fatty acids, intracellular acidosis at -60 mV and mechanical stretch at -40 mV or 40 mV. Furthermore, small interfering RNA (siRNA)-mediated TREK2 knockdown resulted in a slight depolarization from $-19.9mV{\pm}0.8$ (n=116) to $-8.5mV{\pm}1.4$ (n=74) and decreased proliferation of 253J cells, compared to negative control siRNA. 253J cells treated with TREK2 siRNA showed a significant increase in the expression of cell cycle boundary proteins p21 and p53 and also a remarkable decrease in protein expression of cyclins D1 and D3. Taken together, the TREK2 channel is present in bladder cancer cell lines and may, at least in part, contribute to cell cycle-dependent growth.