摘要
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。
ObjectivesTo investigate the regulatory function of tyrosine protein phosphatase non-receptor type 6(PTPN6) on the HERG potassium channel in heart.MethodsThe plasmids of pcDNA3.1-PTPN6-EGFP were constructed by polymerase chain reaction(PCR) technique and transfected or co-transfected with the pcDNA3.1-HERG plasmid into HEK293 cells using Lipofectamine2000. The patch clamp technique was used to document the whole-cell currents of control group(transfected alone with pcDNA3.0-HERG),PTPN6 over expression group(cotransfected with pcDNA3.1-PTPN6-EGFP and pcDNA3.0-HERG),inhibiting group(co-transfected with pcDNA3.1-PTPN6-EGFP and pcDNA3.0-HERG,adding inhibitor of tyrosine protein phosphatase).ResultsThe pcDNA3.1-PTPN6-EGFP plasmid was successfully constructed,the sequence of the cDNA of PTPN6 was correct and in the same reading with EGFP. Green fluorescence representing the fusion protein of PTPN6-EGFP was observed in HEK 293 cells. Both the maximum current density of pulse current[(36.42±2.76)pA/pF]and the maximum current density of tail current[(84.73±7.18)pA/pF]decreased significantly in PTPN6 over expression group,comparing with that[(45.92±3.18)pA/pF,(108.43±7.98)pA/pF]in control group(P<0.05). Comparing with PTPN6 over expression group,both the maximum current density of pulse current[(47.10±2.96)pA/pF]and the maximum current density of tail current[(110.52±7.87) pA/pF] significantly increased in inhibiting group(P<0.05). The time constant of deactivation was significantly higher in PTPN6 over expression group than that in control group[(785.59±90.05)ms vs.(440.7 ± 49.49) ms,P<0.05].ConclusionsOver expression of the PTPN6 decreases HERG potassium channel current,and can be reversed by tyrosine protein phosphatase inhibitor,suggesting that PTPN6 can negatively regulate HERG potassium channel current through dephosphorylating HERG channel.
引文
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