南方医科大学学报

南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (12): 1718-.

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蛋白酪氨酸磷酸酶非受体型12负性调控心脏HERG钾通道电流

林吉进，刘树楷，郑方芳，马青艳，余宏，任莉，沈心远   

  • 出版日期:2013-12-20 发布日期:2013-12-20

Protein tyrosine phosphatase non-receptor type 12 negatively regulates cardiac HERG
channel currents

  • Online:2013-12-20 Published:2013-12-20

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摘要： 目的研究蛋白酪氨酸磷酸酶非受体型12（PTPN12）对心脏HERG钾通道电流的调控作用。方法（1）质粒构建和基因转
染：PCR 技术构建各种质粒，应用脂质体将各种质粒转染HEK293 细胞，经G418 筛选获得稳定表达的细胞株；（2）应用抗
PTPN12 抗体进行Western blotting 分析检测PTPN12 的表达；（3）细胞电生理检测：应用膜片钳技术检测单独表达HERG组
（HEK293/HERG 细胞）、PTPN12 过度表达组（将pCDNA3.1-PTPN12-RFP 转染HEK293/HERG 细胞）、PAO处理组（PTPN12/
HERG组基础上加入抑制剂PAO）、herg 突变组（将pCDNA3.1-PTPN12-RFP 转染HEK293/HERGY327A-Y700A-Y845A细胞
株）的HERG钾通道电流。结果（1）成功构建herg突变质粒和pCDNA3.1-PTPN12-RFP质粒，并获得稳定表达的细胞株；（2）共
转染pCDNA3.1-PTPN12-RFP 质粒的HEK293/HERG细胞在荧光显微镜下可观察到PTPN12-RFP 在细胞中的表达，Western
blotting可检测到PTPN12的表达；（3）PTPN12过度表达组脉冲电流密度明显低于对照组（P<0.01），PAO处理组和herg突变组
电流密度明显高于PTPN12/HERG组（P<0.01）。结论PTPN12对心脏HERG钾通道电流具有明显负性调控作用，其可能机制
是PTPN12减弱了HERG钾通道酪氨酸磷酸化程度。这一发现有助于更深刻理解HERG钾通道调控机制和长QT综合征发病
机制。

Abstract: Objective To study the effect of protein tyrosine phosphatase non-receptor type 12 (PTPN12) in regulating cardiac
HERG channel currents. Methods The plasmids pcDNA3.1-PTPN12-RFP and herg mutant constructed by PCR technique were
transfected into HEK293 cells via Lipofectamine 2000, and the cells stably expressing PTPN12 selected with G418 were
identified by Western blotting with anti-PTPN12 antibody. HERG channel current in cells expressing HERG alone (HEK293/
HERG cells), cells overexpressing PTPN12 (HEK293/HERG cells transfected with pCDNA3.1-PTPN12-RFP), PAO-treated cells
(PTPN12/HERG cells treated with PAO), and herg mutant cells (HEK293/HERGY327A-Y700A-Y845A cells transfected with
pcDNA3.1-PTPN12-RFP) were recorded by patch-clamp technique. Results The plasmids pcDNA3.1-PTPN12-RFP and herg
mutant were successfully constructed, and the stable expressing cell lines were established. Red fluorescence was obversed in
HEK293/HERG cells transfected with pcDNA3.1-PTPN12-RFP, and the protein expression of PTPN12 was detected.
Overexpression of PTPN12 significantly decreased HERG current density in HEK293/HERG cells, and this change was
significantly weakened in the inhibitor group and herg mutant group. Conclusion PTPN12 negatively regulates cardiac HERG
channel cerrent possibly by decreasing the phosphorylation level of HERG tyrosine residues. This finding provides further
insight into the regulatory mechanism of HERG channel and the pathogenesis of long QT syndrome.