Humans ; Potassium Channels, Inwardly Rectifying ; chemistry ; classification ; physiology ; Potassium Channels, Tandem Pore Domain ; chemistry ; classification ; physiology ; Protein Structure, Tertiary

To explore the role of mechanosensitive potassium channel TREK-1, Western blot analysis was used to investigate the expression changes of TREK-1 in left ventricle in acute mechanically stretched heart. Forty Wistar rats were randomly divided into 8 groups (n=5 in each group), subject to single Langendorff perfusion for 0, 30, 60, 120 min and acute mechanical stretch for 0, 30, 60, 120 min respectively. With Langendorff apparatus, an acute mechanically stretched heart model was established. There was no significant difference in the expression of TREK-1 among single Langendorff perfusion groups (P>0.05). As compared to non-stretched Langendorff-perfused heart, only the expression of TREK-1 in acute mechanically stretched heart (120 min) was greatly increased (P<0.05). This result suggested that some course of mechanical stretch could up-regulate the expression of TREK-1 in left ventricle. TREK-1 might play an important role in mechanoelectric feedback, so it could reduce the occurrence of arrhythmia that was induced by extra mechanical stretch.
Feedback ; Heart Ventricles/*metabolism ; Mechanotransduction, Cellular ; Potassium Channels, Tandem Pore Domain/*metabolism ; Random Allocation ; Rats, Wistar ; Stress, Mechanical

This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.
Animals ; Astrocytes ; physiology ; Cells, Cultured ; Gene Silencing ; physiology ; Potassium Channels ; Potassium Channels, Tandem Pore Domain ; genetics ; RNA Interference ; physiology ; RNA, Small Interfering ; genetics ; Rats

To investigate the effect of activating aldehyde dehydrogenase 2 (ALDH2) on TASK-1 two-pore potassium channel in myocardial injury of diabetic rats.
 Methods: Diabetic rats were induced by intraperitoneal injection of streptozotocin (55 mg/kg). The diabetic rats were divided into 4 groups: normal group, diabetes at 4th week (DM4W) group, diabetes at 8th week (DM8W) group, and diabetes at 8th week+low concentration of ethanol intervention (DM8W+EtOH) group. The cardiac function of rats was determined by cardiac ultrasonography. The content of hydroxyproline was detected by ELISA. The appearance of myocardial morphous and positive material were observed by HE and PAS staining. The protein expression of TASK-1 was detected by Western blot. Whole-cell patch clamp technique was used to record the action potential duration at 30% and 90% repolarization (APD30, APD90) and two-pore potassium channel TASK-1 current in rat ventricular myocytes. Meanwhile, according to the sensitive electrophysiological characteristics of the potassium channel to acid and base, whether it is two-port potassium channel TASK-1current can be determined.
 Results: Compared with the N group, end-diastole left ventricular diameter (LVIDd), end-systolic left ventricular diameter (LVIDs), hydroxyproline content, TASK-1 protein expression increased, APD30 and APD90 extend, left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF), and TASK-1 current decreased (all P<0.01) in the DM4W group and the DM8W group. HE staining showed that myocardial cell and fiber arrangement disorder, myocyte hypertrophy, myocardial widened and PAS staining reveals that positive material increased in the DM4W group and the DM8W group. Compared with the DM4W group, these changs are more obvious in DM8W rats (P<0.01 or P<0.05). Compared with the DM8W group, in the DM8W+EtOH group, the left ventricular function was restored, the hydroxyproline content and expression of TASK-1 protein were decreased, the TASK-1 current was increased, and APD30 and APD90 were shortened (all P<0.01). HE staining showed that myocardial cell injury was ameliorate and PAS staining showed decreased deposition of positive substances in the DM8W+EtOH group.
 Conclusion: Activation of aldehyde dehydrogenase 2 by low concentration of ethanol can reduce myocardial injury and fibrosis caused by diabetes, and its mechanism may be related to the changes of the two-por potassium channel TASK-1.
Aldehyde Dehydrogenase, Mitochondrial ; Animals ; Diabetes Mellitus, Experimental ; Heart Diseases ; metabolism ; Myocardium ; Potassium ; Potassium Channels, Tandem Pore Domain ; Rats ; Rats, Sprague-Dawley

BACKGROUND: Volatile general anesthetics have been widely used to produce reversible unconsciousness and analgesia in clinical practice over the last one hundred years, but the basic mechanism of anesthetic action is not yet completely understood. In addition to the well known mechanism of GABAA and glycine channels, accumulating evidence indicates that neuronal baseline K+ channels are also activated by volatile anesthetics. The goal of this study was to test the hypothesis that sevoflurane, one of the newly developed volatile anesthetics, activates baseline potassium channnels in the cerebellar granule neurons of rats. METHODS: Whole cell measurement techniques were performed from cultured cerebellar granule neurons of seven day old male Sprague-Dawley rats using patch clamp techniques to see the effects of two MACs of sevoflurane on baseline K+ channels. Holding potentials were set to 20 mV and collect pulses from 90 to 90 in 10 mV increments of 300 ms duration. The electrode filling solution contained (in mM) 150 KOH, 105 aspartic acid, 3 NaCl, 10 HEPES, 86 glucose, 1 EGTA, 5 MgCl2 (pH 7.4) and standard saline were used as bath solution. The bath contained 150 NaCl, 3 KCl, 10 HEPES, 14 glucose, 1 EGTA, 5 MgCl2 (pH 7.4). RESULTS: Analysis of multiple patch clamp experiments showed the presence of outward-rectifying K+ selective ion channels with a conductance of 1.064 +/- 0.32 nS (n = 10) at depolarized potentials. Cerebellar granule neurons exhibit rapid rising, noninactivating, outward-rectifying currents. These channels are insensitive to conventional K+ channel blockers. Clinically relevant concentrations of sevoflurane (518nM) increased the baseline K+ channel outward currents from the control value by 225% in a standard saline perfusate (n = 10, P < 0.05, paired t-test). Channel activity enhanced during the duration of the exposure period to sevoflurane returned to the baseline activity level quickly upon wash. CONCLUSIONS: These outward-rectifying whole cell I V curves are consistent with the properties of tandem pore K+ channels. Activation of baseline K+ channels in central neurons by two MACs of sevoflurane causes membrane hyperpolarization and increases neuronal input conductances providing an additional inhibitory mechanism that could contribute to the overall central depressant effects of this compound.
Analgesia ; Anesthetics ; Anesthetics, General ; Animals ; Aspartic Acid ; Baths ; Egtazic Acid ; Electrodes ; Glucose ; Glycine ; HEPES ; Humans ; Ion Channels ; Magnesium Chloride ; Male ; Membranes ; Neurons* ; Patch-Clamp Techniques ; Potassium ; Potassium Channels ; Potassium Channels, Tandem Pore Domain* ; Rats ; Rats, Sprague-Dawley* ; Unconsciousness

OBJECTIVETo construct the acid-sensitive potassium hannel-3(TASK3) eukaryotic expression plasmid and to establish a stable SH-SY5Y cell line expressing enhanced green fluorescent protein (EGFP)-tagged TASK3.

METHODSTASK3 coding region was subcloned into pEGFP-N1 plasmid to construct a recombinant vector alled pEGFP-TASK3. The correct recombinant expressing plasmid was transfected with X-feet transfection reagent to SH-SY5Y cells. The cell line stably expressiing EGFP tagged-TASK3 gene was established by screening with antibiotic G418 and fluorescence microscope. The expression and localization of the EGFP tagged-TASK3 fusion protein was detected by Western blot and confocal microscope. Exposure of the SH-SY5Y cell line expressing stably TASK3-eGFP fusion proteins was exposed to different pH media (7.0, 6.7, 6.4, 6.1) for 24 h, the cell viability was assessed with cell counting Kit-8 (CCK-8).

RESULTSAll the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pEGFP-TASK3 was constructed correctly. The stable SH-SY5Y cell line expressing EGFP tagged-TASK3 fusion protein was successfully established. Exposure of the wild type SH-SY5Y cells and the stable SH-SY5Y-GFP tag-TASK3 cell line to different pH media (7.0, 6.7, 6.4, 6.1) for 24 h, the cell viability of two group cells significantly reduced with pH declining, and the difference was statistically significant (P < 0.05). Compared with wild type SH-SY5Y cells, the cell viability of stable SH-SYSY-GFP tag-TASK3 cell line increased significantly with the same pH media, and the difference was statistically significant (P < 0.05).

CONCLUSIONThe eukaryotic expression vector pEGFP-TASK3 is successfully constructed and the cell line stably expressing TASK3-eGFP fusion is established which is important for their fundamental research and potential applications.

Blotting, Western ; Cell Line ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Plasmids ; Polymerase Chain Reaction ; Potassium Channels, Tandem Pore Domain ; genetics ; Transfection

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 specifi c activators of TREK-2 and TRESK may be benefi cial 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.
Acetaminophen ; Amitriptyline ; Analgesics* ; Antidepressive Agents* ; Bupropion ; Citalopram ; Fluoxetine ; Humans ; Ibuprofen ; Membrane Potentials ; Potassium Channels, Tandem Pore Domain ; Sensory Receptor Cells ; Spinal Cord

Animals ; Arachidonic Acid ; pharmacology ; Brain ; metabolism ; Brain Ischemia ; metabolism ; Female ; Humans ; Neuroprotective Agents ; pharmacology ; Ovary ; metabolism ; Potassium Channels ; chemistry ; genetics ; metabolism ; Potassium Channels, Tandem Pore Domain ; chemistry ; genetics ; metabolism ; Riluzole ; pharmacology

OBJECTIVETo investigate the effect of hypoxia on the human pulmonary artery smooth muscle cells two pore domain potassium channels TASK-1 and the regulation of non-receptor tyrosine kinase c-Src in this process.

METHODSThe cultured human pulmonary artery smooth muscle cells (hPASMCs) were divided into: normal group, hypoxia 30 minute group, hypoxia 6 hours group and hypoxia 48 hour group, and hypoxia 48 hour + PP2 group, hypoxia 48 hour + PP3 group, hypoxia 48 hour + bpV group. Flow cytometry was used to analyze the cell cycle, RT-PCR and Western blot technique were carried out to detect the expression changes of TASK-1 mRNA and protein in different groups.

RESULTS(1) Cell Cycle Show: Compared with normal control group, with prolonged hypoxia, the percentages of hPASMCs in S phases of cell cycle were increased. While compared with hypoxia 48 hour group, the percentages of hypoxia 48 hour + PP2 group hPASMCs in S phases of cell cycle were decreased. The expression of TASK-1 mRNA on hPASMCs in acute hypoxia 6 hour group was increased, while the expression of TASK-1 protein on hPASMCs in the acute and chronic hypoxia group was decreased, and the expression of TASK-1 mRNA on hPASMCs in the chronic hypoxia group was decreased; After pre-incubation of a potent and selective inhibitor of the Src family of protein tyrosine kinases PP2, the expression of TASK-1 mRNA and protein in hypoxia 48 hour group was increased, however after pre-incubation of the inhibitor of the Src family of protein tyrosine phosphatase bpV, the expression of TASK-1 protein in hypoxia 48 hour group was decreased.

CONCLUSIONHypoxia promotes human pulmonary artery smooth muscle cell proliferation, and non-receptor tyrosine kinase c-Src may participate in the expression of two pore domain potassium channels TASK-1 regulated by hypoxia. Therefore, we hypothesized that TASK-1 channels and c-Src participatein the acute and chronic hypoxic human pulmonary vasoconstriction.

Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Humans ; Myocytes, Smooth Muscle ; cytology ; Nerve Tissue Proteins ; metabolism ; Potassium Channels, Tandem Pore Domain ; metabolism ; Pulmonary Artery ; cytology ; RNA, Messenger ; Vasoconstriction ; src-Family Kinases ; metabolism

OBJECTIVETo observe the neurological protective effects of progesterone (PROG) on focal cerebral ischemia/reperfusion injury in rats and to explore its possible mechanism.

METHODSOne handred and twenty male SD rats were divided into three groups randomly: sham-operated group, middle cerebral artery occlusion ( MCAO ) group and PROG + MCAO group( n = 40). The right temporary MCAO model was established by the line-embolism method. The PROG + MCAO group rats were according to 8 mg/kg intraperitoneal injection PROG, after that 30 min, the rats were suffered ischemia/reperfusion. After rats were suffered ischemia for 2 h and reperfusion 0, 24, 48, 72 h stress, the nervous functional defect degree were evaluated by longe scoring, and the expression of two-pore domain K channel 3 (TASK3) mRNA in brain tissue were detected by the real-time PCR.

RESULTSPROG (8 mg/kg) could significantly reduced the nervous functional defect degree in rats after ischemia/reperfusion 24, 48, 72 h (P < 0.05). The results of real-time PCR showed that the TASK3 mRNA expression in the brain tissue at all time points significantly decreased in MCAO group compared with sham-operated group (P < 0.05). However, compared with MCAO group, the expression of TASK3 mRNA in brain tissue at all time points dramatically increased in PROG + MCAO group (P < 0.05).

CONCLUSIONPROG can improve the nervous functional defect degree after focal cerebral ischemia/reperfusion injury in rats, and the mechanism might be associated with up-regulating the expression of TASK3 mRNA in brain tissue.

Animals ; Brain ; drug effects ; pathology ; Brain Ischemia ; drug therapy ; Infarction, Middle Cerebral Artery ; Male ; Neuroprotective Agents ; pharmacology ; Potassium Channels, Tandem Pore Domain ; metabolism ; Progesterone ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Reperfusion Injury ; drug therapy