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目的研究长 QT 综合征致病基因之一 KCNH2基因 L413P 和 L559H 突变的致病机制。方法 DNA 定点突变技术构建 L413P 和 L559H 表达载体,脂质体介导法转染人胚胎肾细胞表达。用膜片钳技术记录 KCNH2通道电流变化情况,观察 HERG 蛋白表达和细胞内定位情况。结果电生理学显示 L413P 和 L559H 突变后完全无电流。在转染 L413P 或 L559H 的细胞中只观察到未成熟 HERG 蛋白,而转染 WT 的细胞可观察到成熟和未成熟两种 HERG 蛋白形式。L413P 和 L559H 突变蛋白主要分布在细胞核周围的局部区域,提示突变蛋白滞留于内质网中而 WT 蛋白均匀分布在细胞膜核细胞质中。L413P 或 L559H 与等量野生型质粒共转染后电流相比无显著变化。低温与 E-4031不能纠正 HERG 突变的异常。结论 L413P 和 L559H 突变可引起突变蛋白转运障碍,但基因表达和电生理学结果显示突变对野生型无负显性作用,突变的致病机制可能是单倍体不足。
Objective To study the pathogenesis of KCNH2 L413P and L559H mutations in the gene of long QT syndrome. Methods L413P and L559H expression vectors were constructed by site - directed mutagenesis of DNA. The expression of L413P and L559H was detected by liposome - mediated transfection in human embryonic kidney cells. Patch-clamp technique was used to record the changes of KCNH2 channel currents, HERG protein expression and intracellular localization were observed. Results Electrophysiology showed no current flow after L413P and L559H mutations. Only immature HERG protein was observed in cells transfected with L413P or L559H, whereas both HERG protein forms, mature and immature, were observed in WT transfected cells. The L413P and L559H muteins were localized in the local area around the nucleus, suggesting that the mutein resided in the endoplasmic reticulum and the WT protein was evenly distributed in the cytoplasmic nuclear cytoplasm. L413P or L559H showed no significant change compared with the same amount of wild-type plasmid after co-transfection. Hypothermia and E-4031 do not correct HERG mutation abnormalities. Conclusion The mutations of L413P and L559H may cause the dysfunction of mutein transport, but the gene expression and electrophysiological results show that the mutation has no negative dominant effect on the wild type. The pathogenesis of the mutation may be haploinsufficiency.