KCNJ11基因E23K基因多态对细胞膜电流的影响

Effects of E23K polymorphism in KCNJ11 gene on membrane current

目的:KCNJ11基因E23K多态与心血管疾病、糖尿病等相关联,本实验通过研究人KCNJ11基因外显子处E23K多态对细胞膜电流密度的影响,探讨其与相关疾病关联的机制。方法:普通PCR法扩增KCNJ11基因外显子,重叠延伸PCR法使多态位点G※A突变,基因重组法将KCNJ11基因外显子(分别含23E和23K等位基因)插入pcDNA3.1/CT-GFP真核表达载体,脂质体转染法分别将重组质粒pcDNA3.1-KCNJ11(E)和pcDNA3.1-KCNJ11(K)转入HEK293T细胞。采用全细胞膜片钳技术,检测转染不同质粒的细胞膜电流密度。结果:PCR扩增获得长度为1 173 bp的KCNJ11基因外显子,成功构建pcDNA3.1-KCNJ11(E)和pcDNA3.1-KCNJ11(K)重组表达载体。全细胞膜片钳检测结果显示,两组转染不同质粒的HEK293T细胞表面均检测到正电流和负电流,细胞表面翻转电压均为50mV。两组细胞相比,转染pcDNA3.1-KCNJ11(E)质粒的细胞表面电流明显高于转染pcDNA3.1-KCNJ11(K)质粒的细胞(P<0.05,n=10)。结论:KCNJ11基因外显子E23K多态能导致细胞膜电流发生改变,为进一步研究多态位点与相关疾病的关联机制提供实验基础。

Objective： E23K polymorphism in KCNJll gene is associated with cardiovascular disease and diabetes. In order to explore the mechanism of E23K correlation to related diseases, the effect of E23K polymorphism in KCNJ11 gene on membrane current was investigat- ed. Methods： The exon of KCNJll was obtained by PCR amplification and the G---A mutation was completed by overlap extension PCR. The sequences of KCNJ11 exon contained 23E or 23K was inserted into pcDNA3.1/CT-GFP vector respectively. The recombinant plasmid, pcD- NA3.1-KCNJll（E） and PeDNA3.1-KCNJll（K） , were transfected into HEK293T cells by lipofectamine and the membrane current density was determined by whole-cell patch clamp technique. Results： 1 173 bp sequences of K CNJI 1 gene＇s exon were amplified by PCR and the re- combinant expression plasmid, pcDNA3.1-KCNJ11 （E） and pcDNA3.1-KCNJll （K）, were constructed successful. Positive and negative cur- rents were detected in HEK293T cells transfected with difference plasmid by whole-cell patch clamp technique. Results showed that the re- versed voltage was 50my. The current in HEK293T cells with pcDNA3.1-KCNJll（E） was significantly greater than that with pcDNA3.1-KC- NJll（K） （P 〈 0.05, n = 10）. Conclusion： The polymorphisrn of E23K in exon of KCNJll gene changed the membrane currents in HEK293T cells. It could be an experiment support for the possible mechanism between the locus and related diseases.