本文選題：Brugada綜合征 + KV4.3�。� 參考：《蘇州大學》2012年碩士論文

【摘要】：研究目的：KV4.3和KChIP2亞單位結合構成心臟鉀離子通道，介導快速瞬時外向鉀電流Ito,f，構成心肌細胞動作電位早期復極化過程。Brugada綜合征(Brs)是以心電圖異常和惡性心律失常為特征的遺傳性心臟病。最新研究發(fā)現(xiàn)，Brugada綜合征患者心臟KV4.3通道蛋白羧基末端存在兩個點突變：G600R和L450F，可導致瞬時外向鉀電流上調(diào)、使動作電位穹頂消失。但具體機制未明。因此，本研究旨在探討這兩種BrS相關突變對KV4.3介導電流的影響及其具體作用機制。 方法：我們在KCND3基因上重新構建了這兩個突變并用于在哺乳動物細胞表達，從電生理學、分子生物學角度研究該突變對瞬時外向鉀電流的影響及其具體機制。我們運用全細胞膜片鉗技術研究大鼠KV4.3-G581R(相對應于人類G600R)和KV4.3-L450F對KV4.3介導鉀電流功能的影響；應用Western blot和RT-PCR檢測在與KChIP2共同或單獨表達時，突變對KV4.3蛋白和mRNA表達水平的影響；同時使用免疫熒光定量技術測定在與KChIP2共同或單獨表達時，突變對KV4.3細胞膜表達的影響。 結果：我們發(fā)現(xiàn)，在與KChIP2亞單位共同表達或單獨表達時，這兩種突變都能上調(diào)KV4.3總蛋白表達及細胞膜表達，使鉀電流強度顯著升高。同時，在與KChIP2共同表達時，與KV4.3-WT相比，KV4.3-G581R和KV4.3-L450F都能明顯延緩通道失活。在單獨表達時，KV4.3-G581R和KV4.3-L450F能顯著加速通道失活后恢復速率，并使穩(wěn)態(tài)失活曲線右偏。 結論：鑒于以上數(shù)據(jù)，，我們認為這兩種新發(fā)現(xiàn)的心臟KCND3基因的點突變通過同時增加通道的功能表達和改變其動力學特性這兩種機制上調(diào)心臟快速瞬時外向鉀電流，而KChIP2也在其中發(fā)揮重要作用。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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相關期刊論文 前2條

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2 ;Downregulation of Kυ4.2 and Kυ4.3 channel gene expression in right ventricular hypertrophy induced by monocrotaline in rat[J];Acta Pharmacologica Sinica;2004年02期

本文編號：1961467