The role of human ether a-go-go related gene(hERG) in electrically excitable cells has long been known.hERG currents IKr contribute to the re-polarisation phase 3 of the cardiac action potential. Mutations of this cha...The role of human ether a-go-go related gene(hERG) in electrically excitable cells has long been known.hERG currents IKr contribute to the re-polarisation phase 3 of the cardiac action potential. Mutations of this channel causes long QT syndrome. N629D hERG mutation(GFGN to GFGD) alters the pore selectivity signature sequence.N629D was over-expressed, via adenoviral gene transfer,in car-diomyocytes derived from mouse embryonic stem cells,the "IKr" showed outward rectification and an inward tail current,while WT IKr showed inward rectification and a positive tail current.N629D "IKr" phenotype also altered resting membrane potential and caused arrhythmia.Since hERG was reported to express in early stage of cardiogenesis,the role of the ERG potassium channel in cardiac development was elaborated in an in vivo model of a homozygous. The hERG N629D mutation was introduced into the orthologous mouse gene,mERG,by homologous recombination in mouse embryonic stem cells. N629D/N629D homozygous mutation results in embryonic lethality(died by E11.5).The mutation displayed defect cardiac morphogenesis including altered looping architecture,poorly developed bulbus cordis,and distorted aortic sac and branchial arches. N629D/N629D myocytes from embryonic day 9.5 embryos manifested complete loss of IKr function, depolarized resting potential,prolonged action potential duration(LQT),failure to repolarize,and propensity to oscillatory arrhythmias.Because deletion of transcription factor Hand2 produces apoptosis in similar regions and with a similar final developmental phenotype,Hand2 expression was evaluated. Robust decrease in Hand2 expression was observed in the secondary heart field in N629D/N629D embryos. mERG protein expression in the developing embryonic heart is not homogeneous.The protein expression is exaggerated in the right ventricle and in the outflow tract.N629D/N629D embryos manifest extensive apoptosis particularly in the first branchial arch and the facial region.Given that cells from the branchial arch populate the展开更多
Background Long QT syndrome(LQTS)is a potentially fatal cardiac ion channel disease.Mutations in the gene encoding cardiac hERG potassium channel are the second most common causes of LQTS.Cardiac hERG potassium channe...Background Long QT syndrome(LQTS)is a potentially fatal cardiac ion channel disease.Mutations in the gene encoding cardiac hERG potassium channel are the second most common causes of LQTS.Cardiac hERG potassium channel conducts the rapidly activating delayed rectifier potassium current(Ikr),which is one of the crucial currents in rapid repolarization phase of action potential in human cardiomyocytes.Function of hERG potassium channel is regulated by a variety of signaling pathways,in which phosphorylation and dephosphorylation of tyrosine proteins plays a major role.Previous research has found that non-receptor protein tyrosine phosphatase(PTPN)can interact with hERG potassium channel in cardiac cells.The aims of the present study were to investigate the regulatory effect of protein tyrosine phosphatase non-receptor type 12,11 and 6(PTPN12,PTPN11 and PTPN6)on cardiac hERG potassium channels.Methods HEK-293 cells were transfected with pcDNA3.0-hERG by Lipofectamine 2000 and selected by G418.HEK-293/hERG cells stably expressing hERG protein were then transfected with pcDNA3.1-PTPN12-RFP,pcDNA3.1-PTPN11-EGFP and pcDNA3.1-PTPN6-EGFP,respectively.Forty-eight hours after transfection,immunofluorescence assay and western blot were performed to detect the expression of hERG channel proteins and PTPN proteins.hERG channel currents in hERG alone-expressing group,PTPN12-,PTPN11-and PTPN6-overexpressing groups,as well as inhibitor groups were recorded by patch clamp technique.Results The maximum pulse current densities of PTPN12-,PTPN11-and PTPN6-overexpressing groups were all decreased when compared with hERG alone-expressing group(P<0.05).However,the maximum pulse current densities of inhibitor groups were all increased when compared with PTPN12-,PTPN11-and PTPN6-overexpressing groups,respectively(P<0.05).Conclusions Overexpression of PTPN12,PTPN11 and PTPN6 reduced the current density of hERG potassium channel,while this effect could be reversed by tyrosine phosphatase inhibitors.These results suggested that PTPN12,PTPN11展开更多
目的旨在阐明蛋白酪氨酸磷酸酶非受体型6(tyrosine protein phosphatase non-receptor type 6,PTPN6)是否对心脏HERG钾通道电流具有调控的作用。方法聚合酶链反应(polymerase chain reaction,PCR)技术构建pcDNA3.1-PTPN6-EGFP质粒;应用...目的旨在阐明蛋白酪氨酸磷酸酶非受体型6(tyrosine protein phosphatase non-receptor type 6,PTPN6)是否对心脏HERG钾通道电流具有调控的作用。方法聚合酶链反应(polymerase chain reaction,PCR)技术构建pcDNA3.1-PTPN6-EGFP质粒;应用脂质体Lipofectamine2000将各种质粒转染进入HEK293细胞;应用膜片钳技术分别检测对照组(pcDNA3.0-HERG单独转染HEK293细胞)、PTPN6过度表达组(pcDNA3.0-HERG和pcDNA3.1-PTPN6-EGFP共转染HEK293细胞)以及抑制剂组(pcDNA3.0-HERG和pcDNA3.1-PTPN6-EGFP共转染HEK293细胞,并加入蛋白酪氨酸磷酸酶抑制剂正钒酸钠)的HERG钾通道的脉冲电流最大电流密度、尾电流最大电流密度以及去激活时间常数Tau等。结果成功构建了pcDNA3.1-PTPN6-EGFP质粒,测序结果表明基因序列正确,荧光显微镜下可观察到HEK293细胞中绿色荧光蛋白表达;全细胞膜片钳电生理检测发现,PTPN6过度表达组的脉冲电流最大电流密度[(36.42±2.76)pA/pF]、尾电流最大电流密[(84.73±7.18)pA/pF]均较对照组[(45.92±3.18)pA/pF、(108.43±7.98)pA/pF]显著降低,差异有统计学意义(P<0.05);而抑制剂组脉冲电流最大电流密度、尾电流最大电流密度[(47.10±2.96)pA/pF、(110.52±7.87)pA/pF]均较PTPN6过度表达组明显增大,差异有统计学意义(P<0.05);PTPN6过度表达组失活时间常数Tau[(785.59±90.05)ms]较对照组[(440.7±49.49)ms]明显延长,差异有统计学意义(P<0.05)。结论 PTPN6过度表达能使HERG钾通道的电流密度降低,且这一作用能被酪氨酸磷酸酶抑制剂逆转,提示PTPN6能通过催化HERG钾通道去磷酸化而发挥负性调控HERG钾通道电流的作用。展开更多
文摘The role of human ether a-go-go related gene(hERG) in electrically excitable cells has long been known.hERG currents IKr contribute to the re-polarisation phase 3 of the cardiac action potential. Mutations of this channel causes long QT syndrome. N629D hERG mutation(GFGN to GFGD) alters the pore selectivity signature sequence.N629D was over-expressed, via adenoviral gene transfer,in car-diomyocytes derived from mouse embryonic stem cells,the "IKr" showed outward rectification and an inward tail current,while WT IKr showed inward rectification and a positive tail current.N629D "IKr" phenotype also altered resting membrane potential and caused arrhythmia.Since hERG was reported to express in early stage of cardiogenesis,the role of the ERG potassium channel in cardiac development was elaborated in an in vivo model of a homozygous. The hERG N629D mutation was introduced into the orthologous mouse gene,mERG,by homologous recombination in mouse embryonic stem cells. N629D/N629D homozygous mutation results in embryonic lethality(died by E11.5).The mutation displayed defect cardiac morphogenesis including altered looping architecture,poorly developed bulbus cordis,and distorted aortic sac and branchial arches. N629D/N629D myocytes from embryonic day 9.5 embryos manifested complete loss of IKr function, depolarized resting potential,prolonged action potential duration(LQT),failure to repolarize,and propensity to oscillatory arrhythmias.Because deletion of transcription factor Hand2 produces apoptosis in similar regions and with a similar final developmental phenotype,Hand2 expression was evaluated. Robust decrease in Hand2 expression was observed in the secondary heart field in N629D/N629D embryos. mERG protein expression in the developing embryonic heart is not homogeneous.The protein expression is exaggerated in the right ventricle and in the outflow tract.N629D/N629D embryos manifest extensive apoptosis particularly in the first branchial arch and the facial region.Given that cells from the branchial arch populate the
基金supported by the Key Project of Natural Science Foundation of Guangdong Province of China(No.2017B030311010)Science and Technology Program of Guangzhou,China(No.202002030088)
文摘Background Long QT syndrome(LQTS)is a potentially fatal cardiac ion channel disease.Mutations in the gene encoding cardiac hERG potassium channel are the second most common causes of LQTS.Cardiac hERG potassium channel conducts the rapidly activating delayed rectifier potassium current(Ikr),which is one of the crucial currents in rapid repolarization phase of action potential in human cardiomyocytes.Function of hERG potassium channel is regulated by a variety of signaling pathways,in which phosphorylation and dephosphorylation of tyrosine proteins plays a major role.Previous research has found that non-receptor protein tyrosine phosphatase(PTPN)can interact with hERG potassium channel in cardiac cells.The aims of the present study were to investigate the regulatory effect of protein tyrosine phosphatase non-receptor type 12,11 and 6(PTPN12,PTPN11 and PTPN6)on cardiac hERG potassium channels.Methods HEK-293 cells were transfected with pcDNA3.0-hERG by Lipofectamine 2000 and selected by G418.HEK-293/hERG cells stably expressing hERG protein were then transfected with pcDNA3.1-PTPN12-RFP,pcDNA3.1-PTPN11-EGFP and pcDNA3.1-PTPN6-EGFP,respectively.Forty-eight hours after transfection,immunofluorescence assay and western blot were performed to detect the expression of hERG channel proteins and PTPN proteins.hERG channel currents in hERG alone-expressing group,PTPN12-,PTPN11-and PTPN6-overexpressing groups,as well as inhibitor groups were recorded by patch clamp technique.Results The maximum pulse current densities of PTPN12-,PTPN11-and PTPN6-overexpressing groups were all decreased when compared with hERG alone-expressing group(P<0.05).However,the maximum pulse current densities of inhibitor groups were all increased when compared with PTPN12-,PTPN11-and PTPN6-overexpressing groups,respectively(P<0.05).Conclusions Overexpression of PTPN12,PTPN11 and PTPN6 reduced the current density of hERG potassium channel,while this effect could be reversed by tyrosine phosphatase inhibitors.These results suggested that PTPN12,PTPN11
文摘目的旨在阐明蛋白酪氨酸磷酸酶非受体型6(tyrosine protein phosphatase non-receptor type 6,PTPN6)是否对心脏HERG钾通道电流具有调控的作用。方法聚合酶链反应(polymerase chain reaction,PCR)技术构建pcDNA3.1-PTPN6-EGFP质粒;应用脂质体Lipofectamine2000将各种质粒转染进入HEK293细胞;应用膜片钳技术分别检测对照组(pcDNA3.0-HERG单独转染HEK293细胞)、PTPN6过度表达组(pcDNA3.0-HERG和pcDNA3.1-PTPN6-EGFP共转染HEK293细胞)以及抑制剂组(pcDNA3.0-HERG和pcDNA3.1-PTPN6-EGFP共转染HEK293细胞,并加入蛋白酪氨酸磷酸酶抑制剂正钒酸钠)的HERG钾通道的脉冲电流最大电流密度、尾电流最大电流密度以及去激活时间常数Tau等。结果成功构建了pcDNA3.1-PTPN6-EGFP质粒,测序结果表明基因序列正确,荧光显微镜下可观察到HEK293细胞中绿色荧光蛋白表达;全细胞膜片钳电生理检测发现,PTPN6过度表达组的脉冲电流最大电流密度[(36.42±2.76)pA/pF]、尾电流最大电流密[(84.73±7.18)pA/pF]均较对照组[(45.92±3.18)pA/pF、(108.43±7.98)pA/pF]显著降低,差异有统计学意义(P<0.05);而抑制剂组脉冲电流最大电流密度、尾电流最大电流密度[(47.10±2.96)pA/pF、(110.52±7.87)pA/pF]均较PTPN6过度表达组明显增大,差异有统计学意义(P<0.05);PTPN6过度表达组失活时间常数Tau[(785.59±90.05)ms]较对照组[(440.7±49.49)ms]明显延长,差异有统计学意义(P<0.05)。结论 PTPN6过度表达能使HERG钾通道的电流密度降低,且这一作用能被酪氨酸磷酸酶抑制剂逆转,提示PTPN6能通过催化HERG钾通道去磷酸化而发挥负性调控HERG钾通道电流的作用。
