AIM: To examine the expression of Twik-related K+ channel 1(TREK-1), Twik-related K+ channel 2(TREK-2), and Twik-related arachidonic acid-stimulated K+ channel(TRAAK) in the retina of adult rd1 mice and to detect the ...AIM: To examine the expression of Twik-related K+ channel 1(TREK-1), Twik-related K+ channel 2(TREK-2), and Twik-related arachidonic acid-stimulated K+ channel(TRAAK) in the retina of adult rd1 mice and to detect the protective roles of TREK-TRAAK two-pore-domain K+(K2P) channels against retinal degeneration.METHODS: Twenty-eight-day-old C57BL/6J mice and 28-day-old rd1 mice were used in this study. Retinal protein, retinal RNA, and embedded eyeballs were prepared from these two groups of mice. Real-time quantitative polymerase chain reaction and Western blot analyses were used to assess the gene transcription and protein levels, respectively. Retinal structures were observed using hematoxylin and eosin(H&E) staining. Immunohistochemistry was utilized to observe the retinal localization of TREK-TRAAK channels. Current changes in retinal ganglion cells(RGCs) after activation of TREK-TRAAK channels were examined using a patchclamp technique. RESULTS: Compared with C57BL/6J mice, rd1 mice exhibited significantly higher retinal mRNA and protein expression levels of TREK-1, TREK-2, and TRAAK channels. In both groups, immunohistochemistry showed expression of TREK-TRAAK channels in retinal layers. After addition of the TREK-TRAAK channel agonist arachidonic acid(AA), whole-cell voltage step evoked currents were significantly higher in RGCs from rd1 mice than in RGCs from control C57BL/6J mice, suggesting that TREK-TRAAK channels were opened in RGCs from rd1 mice. CONCLUSION: TREK-TRAAK K2P channels’ expression is increased in adult rd1 mice. AA induced the opening of TREK-TRAAK K2P channels in adult rd1 mice and may thus counterbalance depolarization of RGCs and protect the retina from excitotoxicity. TREK-TRAAK channels may play a protective role against retinal degeneration.展开更多
OBJECTIVE To find that the extracellular cap of a K2P channel can act as a new allosteric site and may serve as a direct drug target.METHODS Molecular biology and cell transfection,electrophysiology,molecular docking,...OBJECTIVE To find that the extracellular cap of a K2P channel can act as a new allosteric site and may serve as a direct drug target.METHODS Molecular biology and cell transfection,electrophysiology,molecular docking,molecular dynamics simulations,virtual screening for TREK1,and depressive-related behavior tests.RESULTS Extracellular domain of TREK1 channel existed a dynamic cavity in the extracellular domain by the method of computations,mutagenesis and electrophysiology.Molecular dynamics simulations suggested that ligand-induced allosteric conformational transitions lead to blockage of the ion conductive pathway.Using virtual screening approach,we identified other inhibitors targeting the extracellular allosteric ligand-binding site of these channels.Overall,our results suggested that the allosteric site at the extracellular cap of the K2P channels might be a promising drug target for these membrane proteins.The TREK1 inhibitor TKDC had significantly faster onset than that of fluoxetine in chronic administration trials,and the study confirms that TREK1 was an important target for the development of rapid antidepressants.CONCLUSION The study is a significant step forward for understanding the function of TREK and for identifying specific inhibitors,which should be of interest to others in the field.展开更多
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-labele...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.展开更多
基金Supported by National Natural Science Foundation of China(No.81271012)
文摘AIM: To examine the expression of Twik-related K+ channel 1(TREK-1), Twik-related K+ channel 2(TREK-2), and Twik-related arachidonic acid-stimulated K+ channel(TRAAK) in the retina of adult rd1 mice and to detect the protective roles of TREK-TRAAK two-pore-domain K+(K2P) channels against retinal degeneration.METHODS: Twenty-eight-day-old C57BL/6J mice and 28-day-old rd1 mice were used in this study. Retinal protein, retinal RNA, and embedded eyeballs were prepared from these two groups of mice. Real-time quantitative polymerase chain reaction and Western blot analyses were used to assess the gene transcription and protein levels, respectively. Retinal structures were observed using hematoxylin and eosin(H&E) staining. Immunohistochemistry was utilized to observe the retinal localization of TREK-TRAAK channels. Current changes in retinal ganglion cells(RGCs) after activation of TREK-TRAAK channels were examined using a patchclamp technique. RESULTS: Compared with C57BL/6J mice, rd1 mice exhibited significantly higher retinal mRNA and protein expression levels of TREK-1, TREK-2, and TRAAK channels. In both groups, immunohistochemistry showed expression of TREK-TRAAK channels in retinal layers. After addition of the TREK-TRAAK channel agonist arachidonic acid(AA), whole-cell voltage step evoked currents were significantly higher in RGCs from rd1 mice than in RGCs from control C57BL/6J mice, suggesting that TREK-TRAAK channels were opened in RGCs from rd1 mice. CONCLUSION: TREK-TRAAK K2P channels’ expression is increased in adult rd1 mice. AA induced the opening of TREK-TRAAK K2P channels in adult rd1 mice and may thus counterbalance depolarization of RGCs and protect the retina from excitotoxicity. TREK-TRAAK channels may play a protective role against retinal degeneration.
基金National Natural Science Foundation of China(3120077181030065+5 种基金8127405531371066013117101101)Ministry of Science and Technology(2013CB91060101)National Science Technology Major Project of China (2012ZX09301-001-062014ZX09102001-005).
文摘OBJECTIVE To find that the extracellular cap of a K2P channel can act as a new allosteric site and may serve as a direct drug target.METHODS Molecular biology and cell transfection,electrophysiology,molecular docking,molecular dynamics simulations,virtual screening for TREK1,and depressive-related behavior tests.RESULTS Extracellular domain of TREK1 channel existed a dynamic cavity in the extracellular domain by the method of computations,mutagenesis and electrophysiology.Molecular dynamics simulations suggested that ligand-induced allosteric conformational transitions lead to blockage of the ion conductive pathway.Using virtual screening approach,we identified other inhibitors targeting the extracellular allosteric ligand-binding site of these channels.Overall,our results suggested that the allosteric site at the extracellular cap of the K2P channels might be a promising drug target for these membrane proteins.The TREK1 inhibitor TKDC had significantly faster onset than that of fluoxetine in chronic administration trials,and the study confirms that TREK1 was an important target for the development of rapid antidepressants.CONCLUSION The study is a significant step forward for understanding the function of TREK and for identifying specific inhibitors,which should be of interest to others in the field.
基金supported by grants from the National Natural Science Foundation of China (No.30971007)the Natural Science Foundation of Hubei Province for Outstanding Young Scholars (No.2010CDA103)the National Basic Research Program(No.2011CB504403)
文摘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.
