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  

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

作　　者：林吉进[1,2] 刘树楷[1,2] 郑方芳[1,2] 马青艳[1,2] 余宏[1,2] 任莉[1,2] 沈心远 

机构地区：[1]广东省心血管病研究所 [2]广东省人民医院心内科,广东广州510080

出　　处：《南方医科大学学报》2013年第12期1718-1722,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(81070151);广东省自然科学基金(S2012010009324)~~

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。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.

关 键 词：长QT综合征 HERG钾通道 蛋白酪氨酸磷酸酶非受体型12 膜片钳技术 

分 类 号：R331[医药卫生—人体生理学]