Progress in understanding the central nervous system pharmacology of the two-pore domain potassium channel TREK-1 / 药学学报

Two-pore domain potassium channels (K2P) make up a subfamily of potassium channels discovered in the 1990s, and TREK-1 is the most widely studied subtype of K2P. TREK-1 is widely expressed in the body and especially in the central nervous system, where its main role is to control cell excitability and maintain the membrane potential below the depolarization threshold. It thereby participates in regulating various physiological and pathological processes. TREK-1 is also a potential drug target in many diseases. It is known that many marketed drugs can affect the function of TREK-1, but currently there are no specific TREK-1 modulators or drugs. We review the structure, distribution and regulation of TREK-1 and focus on recent progress in understanding the pharmacology of TREK-1 and its role in neuroprotection, depression, anesthesia and epilepsy. The research status of TREK-1 modulators is discussed.

Effects of clostridium butyricum on intestinal barrier function in food allergic mice / 重庆医学

Objective To investigate that butyrate-producing probiotic clostridium butyricum improves the intestinal epithelial barrier function in food allergic mice by regulating TWIK-related potassium channel-1 (Trek1) expression in intestinal epithelial cells.Methods An intestinal allergy mouse model was created,then the model construction effect was verified by detecting the related indicators by ELISA,flow cytometer.The change of small intestinal tissue permeability was detected by the Ussing chambers.The Trek1 expressions in mouse jejunum tissue in control group and allergy group were detected by Western blot and immunofluorescent method;in the Transwell system,T84 cells were used to establish epithelial cellular monolayer for exposing to the allergic mediators,the Trek1 mRNA and protein expression were detected by qRT-PCR and Western blot.Then the allergic mice were grouped and treated by different methods including normal saline,SIT,SIT/SB,SB,SIT/CB,CB,SIT/CB/Spadin,the expression of mice intestinal Trek1,intestinal barrier function and allergic reaction indicators were detected.Results Compared with the control group,the small intestinal Trek1 protein and tissue expression level in the food allergic mice were significantly decreased,the intestinal mucosal permeability was significantly increased,the differences were statistically significant(P＜0.05);after T84 cells exposing to the allergic mediators,Trek1 mRNA and protein expression were significantly decreased(P＜0.05),but adding p38 inhibitor in advance could antagonize this change;the single use of SIT,clostridium butyricum and sodium butyrate could increase the intestinal Trek1 expression in food allergic mice,and alleviated the allergic reaction(P＜0.05),whereas the combined use of SIT and clostridium butyricum or sodium butyrate had more significant effect(compared with the SIT group,P＜0.05),moreover could significantly decrease small intestinal mucosal permeability and improved the intestinal barrier function(P＜0.05).Conclusion Sodium butyrate or butyrate-producing probiotic clostridium butyricum can restore the intestinal barrier function,alleviates the allergic reaction and strengthens the SIT curative effect in allergic mice by increasing Trek1 expression.

The improvement effect of TREK-1 potassium channel blockade on the behavior of depression model rats / 中华行为医学与脑科学杂志

Objective To study the effect of TREK-1 potassium channel blocker on the behavior in rats with depression.Methods Forty-eight healthy adult male Sprague Dawley (SD) rats were divided into 6 groups as following:control + saline (CON group),control + fluoxetine (CON + FLU group),control + SID1900 (CON+SID group),CUMS + saline (CUMS group),CUMS + fluoxetine (CUMS+FLU group) and CUMS + SID1900 (CUMS+SID group) with 8 in each group.Isolated living conditions combining chronic unpredicted mild stress (CUMS) were used to establish depression model in rats.Four weeks after modeling,fluoxetine 10 mg· kg-1,SID1900 5.1 mg · kg-1 and 0.9％ sodium chloride were given by intraperitoneal injection respectively.Body weight and behavioral performance of rats in several experiment paradigms were measured after drug administration.The behavioral paradigms included sucrose preference test(SPT),open field test(OFT) and forced swimming test(FST).Results Drug administration had no significant effect on body weight and behavioral performance in non stress rats (P＞0.05),however,after chronic unpredicted mild stress the body weight,percentage of sucrose consumption,movement distances and rearing times in CUMS group were decreased dramatically contrast to CON group,but the immobility duration was increased significantly (all P＜0.001).After treatment with drug for 14 days,the sucrose consumption percentage(62.03± 6.99) ％) and rearing times (18.57 ± 6.37) in CUMS+SID group were increased contrast to C UMS group ((45.46± 15.54) ％,(5.83±3.06)),while the immobility time((123.57±26.73) s) was decreased compared with that in CUMS group((174.33±40.68) s).When drug administration for 28 days,the sucrose consumption percentage((79.64± 11.37) ％),the total distance as well as the rearing times ((13.18 ± 3.17) m,(19.33±3.33)) within OFT in CUMS+FLU group were increased in comparison with CUMS group((48.06± 17.10) ％,(4.45±3.69) m,(5.17± 2.99)),and the immobility duration ((97.83± 18.97) s) in CUMS+FLU group was shorter than that ((194.83±37.97) s) in CUMS group(P＜0.05).Notably,the immobility time in CUMS+SID group ((44.29± 14.30)s) was shortened obviously compared with that in CUMS+FLU group ((97.83± 18.97)s) (P＜0.01).Conclusion Blockade of TREK-1 potassium channel can ameliorate the depression-like behavior rapidly in rats.

Construction of functional and concatenated dimers of two-pore-domain potassium channel TREK-1 / 军事医学

Objective To explore the feasibility of adding a flexible linker between two-pore-domain potassium channel TREK-1 (TWIK related K + channel 1)monomers to construct a tandem-linked dimer.Methods PCR was used to add a flexible linker between the two TREK-1 monomers.The cRNA obtained from in vitro transcription using the above vector was injected into Xenopus oocytes.After 24 -48 h,currents were recorded from these oocytes using a two-electrode voltage clamp.The effects of extracellular Ba2 + and pH on TdTREK-1 were observed and compared with those of native dimeric TREK-1.Results The tandem-linked dimeric TdTREK-1 was highly expressed in Xenopus oocytes.The currents through these channels were inhibited by extracellular Ba2 +and acidification.Furthermore,the responsiveness of the concatenated dimers to these extracellular stimuli was similar to that of native dimers.Conclusion Adding a flexible linker between the two monomers to construct the tandem-linked dimer does not affect the expression and gating properties of TREK-1, suggesting that the method be feasible.Such a method will allow the manipulation of a single subunit,which will help basis study the structure and function of TREK-1.

Isoflurane Preconditioning Induces Neuroprotection by Up-Regulation of TREK1 in a Rat Model of Spinal Cord Ischemic Injury

This study aimed to explore the neuroprotection and mechanism of isoflurane on rats with spinal cord ischemic injury. Total 40 adult male Sprague-Dawley rats were divided into the four groups (n=10). Group A was sham-operation group; group B was ischemia group; group C was isoflurane preconditioning group; group D was isoflurane preconditioning followed by ischemia treatment group. Then the expressions of TWIK-related K⁺ channel 1 (TREK1) in the four groups were detected by immunofluorescent assay, real time-polymerase chain reactions (RT-PCR) and western blot. The primary neurons of rats were isolated and cultured under normal and hypoxic conditions. Besides, the neurons under two conditions were transfected with green fluorescent protein (GFP)-TREK1 and lentivirual to overexpress and silence TREK1. Additionally, the neurons were treated with isoflurane or not. Then caspase-3 activity and cell cycle of neurons under normal and hypoxic conditions were detected. Furthermore, nicotinamide adenine dinucleotide hydrate (NADH) was detected using NAD+/NADH quantification colorimetric kit. Results showed that the mRNA and protein expressions of TREK1 increased significantly in group C and D. In neurons, when TREK1 silenced, isoflurane treatment improved the caspase-3 activity. In hypoxic condition, the caspase-3 activity and sub-G1 cell percentage significantly increased, however, when TREK1 overexpressed the caspase-3 activity and sub-G1 cell percentage decreased significantly. Furthermore, both isoflurane treatment and overexpression of TREK1 significantly decreased NADH. In conclusion, isoflurane-induced neuroprotection in spinal cord ischemic injury may be associated with the up-regulation of TREK1.

Enhanced Expression of TREK-1 Is Related with Chronic Constriction Injury of Neuropathic Pain Mouse Model in Dorsal Root Ganglion

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.

Effects of fluoxetine on protein expression of potassium ion channels in the brain of chronic mild stress rats

The purpose of this study is to investigate the expression of major potassium channel subtypes in the brain of chronical mild stress (CMS) rats and reveal the effects of fluoxetine on the expression of these channels. Rats were exposed to a variety of unpredictable stress for three weeks and induced anhedonia, lower sucrose preference, locomotor activity and lower body weight. The protein expressions were determined by Western blot. CMS significantly increased the expression of Kv2.1 channel in frontal cortex but not in hippocampus, and the expression level was normalized after fluoxetine treatment. The expression of TREK-1 channel was also obviously increased in frontal cortex in CMS rats. Fluoxetine treatment might prevent this increase. However, the expression of Kv3.1 and Kv4.2 channels was considerably decreased in hippocampus after CMS, and was not affected by fluoxetine. These results suggest that different subtypes of potassium channels are associated with the pathophysiology of depression and that the therapeutical effects of fluoxetine may relate to Kv2.1 and TREK-1 potassium channels.

The antidepressant and neuroprotective effect mediated by TREK-1 potassium channel / 中华行为医学与脑科学杂志

Objective Recently, the role of TREK-1 in anti-depression and neuroprotection was demonstrated definitely.In this paper,a systematic review about the functional structure of TREK-1 as well as the underlying mechanisms which involved in depression resistance and nerve protection was made for purpose of providing new ideas for the treatment of clinical diseases.Methods The electronic search of Medline/PubMed,CNKI,CBM and Wanfang Data were performed to collect the introduction of TREK-1 and the studies of depression and nerve protection in which TREK-1 participated from inception of each database to October 2015 ,besides the relevant references of the included studies were also retrieved.Results 30 studies were included in this paper.14 studies were about the interpretation of TREK-1's structure, distribution and the regulatory factors, and the number of researches regarding the involvement of TREK-1 in anti-depression and neuroprotection was 13 and 7 respectively.Based on these studies,it was easily to conclude that inhibition or blocking of TREK-1 channels can produce obvious antidepressant effect, however, activation of TREK-1 channels will induce the dilation of local cerebral vascular and silence hyperactive neurons.Conclusion Some achievements about the antidepressant and neuroprotective effect of TREK-1 channels in vivo have been made gratifyingly,thus future research should focus on exploring the best TREK-lactivation ratio in different brain regions to gain the best balance between anti-depression and neuroprotection.

Functional expression of mechanosensitive two-pore domain potassium channel in human bladder carcinoma cells / 동물의과학연구지

Bladder cancer is a common cancer in smoking men and may correlate with mechanosensitive potassium channels because the urinary bladder is a stretch sensing organ. Two-pore K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to play a critical role in both cell apoptosis and tumorigenesis. Of the channels, TREK1 can be activated by many physiological stimuli, including polyunsaturated fatty acids, and intracellular pH, hypoxia, and neurotransmitters. Here we attempted to determine whether TREK1 is functionally expressed in bladder cancer 253J cells. K2P channels, including TREK1, TREK2, TASK1, TASK3, and TWIK1, were quantified in cultured human bladder cancer 253J cells using real time quantitative RT-PCR (qRT-PCR) analysis. Among them, TREK1-like channel was recorded at a single channel level using the patch-clamp technique. The TREKl-like channel, with single-channel conductance of ~90 pS at -80 mV, was recorded in symmetrical 150 mM KCl using an excised inside-out patch configuration. The current-voltage relationships were linear and were insensitive to tetraethylammonium. The channel was activated by membrane stretch, free fatty acids, and intracellular acidosis. These results with electrophysiological properties resemble to those of K2P channel, for instance, TREK1. Therefore, we conclude that TREK1 channel is functionally present in bladder cancer 253J cells.