Brugada综合征相关基因KCND3突变通过上调Kv4.3膜蛋白表达及影响通道动力学特性增加心脏快速瞬时外向钾电流

发布时间：2018-05-31 22:09

本文选题：Brugada综合征 + KV4.3　；参考：《苏州大学》2012年硕士论文

【摘要】：研究目的：KV4.3和KChIP2亚单位结合构成心脏钾离子通道，介导快速瞬时外向钾电流Ito,f，构成心肌细胞动作电位早期复极化过程。Brugada综合征(Brs)是以心电图异常和恶性心律失常为特征的遗传性心脏病。最新研究发现，Brugada综合征患者心脏KV4.3通道蛋白羧基末端存在两个点突变：G600R和L450F，可导致瞬时外向钾电流上调、使动作电位穹顶消失。但具体机制未明。因此，本研究旨在探讨这两种BrS相关突变对KV4.3介导电流的影响及其具体作用机制。方法：我们在KCND3基因上重新构建了这两个突变并用于在哺乳动物细胞表达，从电生理学、分子生物学角度研究该突变对瞬时外向钾电流的影响及其具体机制。我们运用全细胞膜片钳技术研究大鼠KV4.3-G581R(相对应于人类G600R)和KV4.3-L450F对KV4.3介导钾电流功能的影响；应用Western blot和RT-PCR检测在与KChIP2共同或单独表达时，突变对KV4.3蛋白和mRNA表达水平的影响；同时使用免疫荧光定量技术测定在与KChIP2共同或单独表达时，突变对KV4.3细胞膜表达的影响。结果：我们发现，在与KChIP2亚单位共同表达或单独表达时，这两种突变都能上调KV4.3总蛋白表达及细胞膜表达，使钾电流强度显著升高。同时，在与KChIP2共同表达时，与KV4.3-WT相比，KV4.3-G581R和KV4.3-L450F都能明显延缓通道失活。在单独表达时，KV4.3-G581R和KV4.3-L450F能显著加速通道失活后恢复速率，并使稳态失活曲线右偏。结论：鉴于以上数据，，我们认为这两种新发现的心脏KCND3基因的点突变通过同时增加通道的功能表达和改变其动力学特性这两种机制上调心脏快速瞬时外向钾电流，而KChIP2也在其中发挥重要作用。
[Abstract]:Objective to construct a potassium channel in the heart by combining the subunit of: KV4.3 with KChIP2. Rapid transient outward potassium current Itof, which constitutes the early repolarization process of cardiomyocyte action potential. Brugada syndrome (Brs) is a hereditary heart disease characterized by abnormal electrocardiogram and malignant arrhythmia. Two point mutations at the carboxyl end of the KV4.3 channel protein in the heart of patients with Brugada syndrome have been found: G600R and L450F, which can cause transient outward potassium current upregulation and the disappearance of the action potential dome. However, the specific mechanism is not clear. Therefore, the purpose of this study was to investigate the effects of these two BrS related mutations on KV4.3 mediated currents and their specific mechanisms. Methods: we reconstructed these two mutations on the KCND3 gene and used them for expression in mammalian cells. The effects of these mutations on transient outward potassium currents and their specific mechanisms were investigated from electrophysiology and molecular biology. We used whole-cell patch clamp technique to study the effects of rat KV4.3-G581R (corresponding to human G600R) and KV4.3-L450F on KV4.3 mediated potassium current function, and to detect the effect of mutation on KV4.3 protein and mRNA expression level by Western blot and RT-PCR. At the same time, immunofluorescence quantitative technique was used to detect the effect of mutation on the expression of KV4.3 cell membrane when it was co-expressed with KChIP2 or alone. Results: we found that both of these mutations could up-regulate the expression of KV4.3 total protein and cell membrane and increase the potassium current intensity when co-expressed with or without KChIP2 subunit. At the same time, when co-expressed with KChIP2, both KV4.3-G581R and KV4.3-L450F could significantly delay channel inactivation compared with KV4.3-WT. When expressed alone, KV4.3-G581R and KV4.3-L450F can significantly accelerate the recovery rate after channel inactivation, and make the steady-state inactivation curve deviate to the right. Conclusion: in view of the above data, we believe that the point mutations of these two newly discovered cardiac KCND3 genes up-regulate the transient outward potassium currents by simultaneously increasing the functional expression of the channels and changing their dynamic characteristics. And KChIP2 also plays an important role in it.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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