HERG錯(cuò)義突變A422T和H562P導(dǎo)致LQT綜合征的細(xì)胞分子機(jī)制
發(fā)布時(shí)間：2018-08-07 14:13
【摘要】：HERG鉀通道電流是心肌細(xì)胞動(dòng)作電位的重要組分,在3期復(fù)極化晚期發(fā)揮關(guān)鍵作用。藥物的阻斷作用或HERG基因突變?cè)斐稍撾娏鳒p少,可分別導(dǎo)致獲得性LQTS和遺傳性LQTS (LQT2)。就LQT2而言,目前已發(fā)現(xiàn)的突變位點(diǎn)有200多個(gè),但致病機(jī)制各不相同。 A422T和H562P是從兩個(gè)不同家庭LQTS病人篩選到的錯(cuò)義突變位點(diǎn),電生理分析結(jié)果顯示,這兩種不同位點(diǎn)突變通道電流明顯減小,功能缺失,但具體的發(fā)生機(jī)制是何,尚不清楚。本課題利用膜片鉗技術(shù)結(jié)合細(xì)胞分子生物學(xué)技術(shù),研究分析A422T和H562P突變導(dǎo)致HERG鉀通道功能缺失的機(jī)制,并探討不同方法的挽救效果,為理解通道蛋白運(yùn)輸缺陷導(dǎo)致HERG鉀通道功能缺失、造成LQT2的細(xì)胞分子機(jī)制提供詳實(shí)的實(shí)驗(yàn)依據(jù),同時(shí)為LQTS疾病的臨床治療提供新視點(diǎn)。 主要結(jié)果如下： 1.首先,利用全細(xì)胞膜片鉗技術(shù),對(duì)分別表達(dá)A422T和H562P突變通道的HEK 293細(xì)胞進(jìn)行檢測(cè),結(jié)果發(fā)現(xiàn)兩種錯(cuò)義突變均可導(dǎo)致HERG鉀通道電流幅度減少,電壓依賴性消失,說明A422T和H562P存在不同程度的功能缺失。Western blot實(shí)驗(yàn)結(jié)果顯示,A422T和H562P通道的155 kDa成熟蛋白組分消失,提示突變蛋白存在轉(zhuǎn)運(yùn)障礙。這一結(jié)果進(jìn)一步在免疫細(xì)胞化學(xué)檢測(cè)實(shí)驗(yàn)得到證實(shí),表現(xiàn)在A422T與H562P突變通道蛋白膜分布減少,胞質(zhì)內(nèi)蛋白增加,且與內(nèi)質(zhì)網(wǎng)共定位,說明兩種突變通道蛋白由于轉(zhuǎn)運(yùn)障礙而被滯留在內(nèi)質(zhì)網(wǎng)內(nèi)。 2.其次,將WT與A422T或H562P的質(zhì)粒共轉(zhuǎn)HEK 293細(xì)胞,利用全細(xì)胞膜片鉗技術(shù)及western blot檢測(cè)的結(jié)果顯示,雜合型通道的電流及成熟蛋白表達(dá)均顯著少于WT,并且隨著轉(zhuǎn)染突變體質(zhì)粒量的增加,通道功能和蛋白表達(dá)受損加重,說明A422T、H562P對(duì)WT的電流及蛋白表達(dá)有濃度依賴的負(fù)顯性抑制作用。進(jìn)一步對(duì)A422T/WT(轉(zhuǎn)染時(shí)質(zhì)粒的量為1：1,模擬病人常見雜合基因型)、H562P/WT(轉(zhuǎn)染時(shí)質(zhì)粒的量為1：1,模擬病人常見雜合基因型)的電生理特性進(jìn)行檢測(cè)分析,其結(jié)果表明,除成熟蛋白表達(dá)減少外,雜合型通道的動(dòng)力學(xué)與WT相比也有改變,A422T/WT主要表現(xiàn)在激活過程減慢,而H562P/WT表現(xiàn)在失活過程加快。而與A422T/WT相比,H562P/WT具有更快激活,更快失活的特點(diǎn),這將使通道得開放時(shí)間縮短,進(jìn)而顯著影響動(dòng)作電位復(fù)極化的鉀離子流。這種動(dòng)力學(xué)的改變是導(dǎo)致雜合型通道功能缺陷的另一原因。 3.進(jìn)一步利用不同方法對(duì)A422T、H562P突變通道進(jìn)行處理,比較其對(duì)電流及蛋白表達(dá)的挽救作用,分析兩突變體的結(jié)構(gòu)及轉(zhuǎn)運(yùn)障礙的分子機(jī)制。在低溫或E4031處理?xiàng)l件下,A422T突變通道的功能和蛋白轉(zhuǎn)運(yùn)均可得到顯著改善,不但再次證明A422T突變通道蛋白轉(zhuǎn)運(yùn)障礙發(fā)生于內(nèi)質(zhì)網(wǎng),并且說明該突變通道蛋白可形成基本的四聚體結(jié)構(gòu)及孔道結(jié)構(gòu),系一種錯(cuò)誤折疊不嚴(yán)重的突變蛋白；相反,兩種處理對(duì)H562P突變通道卻無效,說明其蛋白折疊錯(cuò)誤嚴(yán)重,無法被常用方法挽救。 在過表達(dá)Hsp70和/或Hsp90的情況下,Co-IP檢測(cè)證實(shí)A422T或H562P與Hsp70和Hsp90三者之間存在相互作用,并且與野生型HERG鉀通道蛋白相比,突變通道蛋白與Hsp70或Hsp90的結(jié)合量也不同；過表達(dá)Hsp70和Hsp90對(duì)A422T突變通道電流有部分挽救作用,而對(duì)蛋白表達(dá)沒有顯著影響；這種挽救作用存在于單獨(dú)過表達(dá)Hsp90而不是Hsp70的條件下,雷公藤紅素——Hsp90的抑制劑可取消其挽救作用。這些說明Hsp70、Hsp90均參與HERG鉀通道蛋白的轉(zhuǎn)運(yùn)過程,但Hsp90對(duì)A422T的蛋白轉(zhuǎn)運(yùn)更為重要。對(duì)H562P突變通道而言,盡管與Hsp70和Hsp90存在相互作用,但無論過表達(dá)Hsp70和/或Hsp90,對(duì)其功能和蛋白表達(dá)都無明顯影響,進(jìn)一步說明兩種不同位置的錯(cuò)義突變,影響其功能的機(jī)制存在差異。H562P突變無法被內(nèi)質(zhì)網(wǎng)質(zhì)量控制系統(tǒng)糾正。 在適度低氧刺激(2%O2作用24hr)下,對(duì)WT通道電流及蛋白成熟過程均有明顯促進(jìn)作用,也可使A422T突變通道功能部分恢復(fù),但對(duì)H562P無影響。提示適當(dāng)?shù)膬?nèi)質(zhì)網(wǎng)應(yīng)激(ER stress)可以促進(jìn)HERG鉀通道蛋白的轉(zhuǎn)運(yùn)、成熟。 4.最后,利用反轉(zhuǎn)錄PCR實(shí)驗(yàn)技術(shù)檢測(cè)XBP1的剪切情況,了解兩突變體是否在細(xì)胞內(nèi)過度堆積,引起ER stress。實(shí)驗(yàn)結(jié)果表明A422T和H562P突變通道蛋白雖然阻滯于內(nèi)質(zhì)網(wǎng)中,但并未過多堆積,也未造成ER stress,而是及時(shí)進(jìn)入泛素-蛋白酶體途徑進(jìn)行降解。 結(jié)論： 1.HERG錯(cuò)義突變A422T和H562P使通道蛋白轉(zhuǎn)運(yùn)障礙并阻滯于內(nèi)質(zhì)網(wǎng),是導(dǎo)致通道功能缺失并引起LQTS (LQT2)的細(xì)胞分子機(jī)制； 2.A422T突變體是錯(cuò)誤折疊不嚴(yán)重的蛋白,可形成四聚體結(jié)構(gòu),在低溫、藥物、分子伴侶或低氧處理后可恢復(fù)部分功能； 3.H562P突變體,尚沒有有效的方法挽救其功能�？赡苁怯捎贖562P位于S5跨膜片段,位于孔道內(nèi)部,位置關(guān)鍵且隱蔽,難以糾正。
[Abstract]:HERG potassium channel current is an important component of the action potential of cardiac myocytes, which plays a key role in the late phase of the 3 repolarization. The blocking action of the drug or the mutation of HERG gene causes the decrease of the current, which can lead to acquired LQTS and hereditary LQTS (LQT2). As far as LQT2 is concerned, there are now more than 200 mutation sites, but the pathogenic mechanisms are different. Same.
A422T and H562P are missense mutation sites selected from two different family LQTS patients. Electrophysiological analysis shows that the current of these two different loci mutations is obviously reduced and the function is missing, but what is the specific mechanism is not clear. This subject uses patch clamp technique and cell molecular biology technology to study and analyze A422T and analysis. H562P mutation leads to the mechanism of HERG potassium channel dysfunction, and explores the effect of different methods. It provides a detailed experimental basis for understanding the loss of channel protein transport caused by HERG potassium channel function loss, resulting in the molecular mechanism of LQT2 cells, and provides new view for the clinical treatment of LQTS disease.
The main results are as follows:
1. first, the whole cell patch clamp technique was used to detect HEK 293 cells expressing A422T and H562P mutation channels respectively. The results showed that the two kinds of missense mutations could lead to the decrease of the current amplitude and the voltage dependence of the HERG potassium channel, indicating that the.Western blot experimental results of A422T and H562P have different degrees of functional deletion.Western blot, and A422T and H5 are shown. The 155 kDa mature protein components of the 62P channel disappeared, suggesting that the mutant protein had a transport barrier. This result was further confirmed in the immunocytochemical test, showing a decrease in the distribution of protein membrane in the mutant channel of A422T and H562P, the increase in the cytoplasmic protein, and the co loci with the endoplasmic reticulum, indicating that the two mutant channel proteins are due to the transport barrier. It was stuck in the endoplasmic reticulum.
2. secondly, the plasmids of WT and A422T or H562P were converted to HEK 293 cells. The results of whole cell patch clamp technique and Western blot detection showed that the current and mature protein expression of the heterozygous channel were significantly less than that of WT, and the channel power and protein expression were aggravated with the increase of the particle quantity of the transfection mutation, indicating A422T and H562P to WT. The current and protein expression has a concentration dependent negative dominance inhibition effect. Further analysis of the electrophysiological characteristics of A422T/WT (transfected plasmid 1:1, simulated patient's common heterozygous genotype), H562P/WT (transfected plasmids as 1:1, simulated patient common heterozygous genotypes), the results showed that the expression of the mature protein was expressed. In addition, the kinetics of heterozygous channel changes compared with that of WT, and A422T/WT is mainly manifested in the slow activation process and the H562P/WT performance is accelerated in the inactivation process. Compared with A422T/WT, H562P/WT has the characteristics of faster activation and faster inactivation, which will shorten the opening time of the channel, and thus significantly affect the potassium ionization of the action potential repolarization. This change in kinetics is another reason for the dysfunction of heterozygous channels.
3. further use different methods to treat A422T, H562P mutation channel, compare the salvage effect of the current and protein expression, analyze the structure of the two mutant and the molecular mechanism of the transport barrier. Under the condition of low temperature or E4031 treatment, the function of the A422T mutation channel and the transfer of protein can be significantly improved, not only to prove A422T again, but not only again. The mutant channel protein transport barrier occurs in the endoplasmic reticulum and indicates that the mutant channel protein can form the basic four polymer structure and pore structure, which is a wrong folding and not serious mutation protein. On the contrary, the two treatments are invalid for the H562P mutation channel, indicating that the protein folding error is serious and can not be saved by the common methods.
In the case of overexpression of Hsp70 and / or Hsp90, Co-IP detection confirmed the interaction between A422T or H562P and Hsp70 and Hsp90 three, and the binding amount of the mutant channel protein to Hsp70 or Hsp90 is different compared with the wild type HERG potassium channel protein, and the overexpression of Hsp70 and Hsp90 has partially saved the current of the mutation channel. There is no significant effect of protein expression; this salvage effect exists in the condition of single overexpression of Hsp90 rather than Hsp70. The inhibitor of tripterin - Hsp90 cancels its salvage effect. These indicate that Hsp70 and Hsp90 are all involved in the transport process of HERG potassium channel protein, but Hsp90 is more important for the protein transport of A422T. In spite of the interaction with Hsp70 and Hsp90, the expression of Hsp70 and / or Hsp90 has no significant effect on its function and protein expression, further explaining the missense mutations in two different locations, and the mechanism that affects its function can not be corrected by the quality control system of the endoplasmic reticulum.
Under the moderate hypoxia stimulation (2%O2 action 24hr), it has a significant effect on the current and protein maturation of the WT channel, but also can restore the functional part of the A422T mutation channel, but has no effect on H562P. It suggests that appropriate endoplasmic reticulum stress (ER stress) can promote the transport and maturation of the egg white in the potassium channel of HERG.
4. finally, using the reverse transcriptional PCR test technique to detect the shear condition of XBP1, to understand whether the two mutants are overstacked in the cells and cause the ER stress. experimental results to show that the A422T and H562P mutant channel proteins are blocked in the endoplasmic reticulum, but do not accumulate too much and do not cause ER stress, but enter the ubiquitin proteasome pathway in time. Degrading.
Conclusion:
1.HERG missense mutations A422T and H562P cause the barrier of channel protein transport and block in the endoplasmic reticulum, which is a cellular mechanism that causes loss of channel function and causes LQTS (LQT2).
2. The A422T mutant is a non-misfolded protein that can form a tetramer structure and restore some of its functions at low temperatures, drugs, molecular chaperones or hypoxia.
The 3.H562P mutant has no effective method to save its function. It may be because H562P is located in the S5 transmembrane fragment, located inside the channel, and the position is key and concealed, and it is difficult to correct.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346
本文編號(hào)：2170282
[Abstract]:HERG potassium channel current is an important component of the action potential of cardiac myocytes, which plays a key role in the late phase of the 3 repolarization. The blocking action of the drug or the mutation of HERG gene causes the decrease of the current, which can lead to acquired LQTS and hereditary LQTS (LQT2). As far as LQT2 is concerned, there are now more than 200 mutation sites, but the pathogenic mechanisms are different. Same.
A422T and H562P are missense mutation sites selected from two different family LQTS patients. Electrophysiological analysis shows that the current of these two different loci mutations is obviously reduced and the function is missing, but what is the specific mechanism is not clear. This subject uses patch clamp technique and cell molecular biology technology to study and analyze A422T and analysis. H562P mutation leads to the mechanism of HERG potassium channel dysfunction, and explores the effect of different methods. It provides a detailed experimental basis for understanding the loss of channel protein transport caused by HERG potassium channel function loss, resulting in the molecular mechanism of LQT2 cells, and provides new view for the clinical treatment of LQTS disease.
The main results are as follows:
1. first, the whole cell patch clamp technique was used to detect HEK 293 cells expressing A422T and H562P mutation channels respectively. The results showed that the two kinds of missense mutations could lead to the decrease of the current amplitude and the voltage dependence of the HERG potassium channel, indicating that the.Western blot experimental results of A422T and H562P have different degrees of functional deletion.Western blot, and A422T and H5 are shown. The 155 kDa mature protein components of the 62P channel disappeared, suggesting that the mutant protein had a transport barrier. This result was further confirmed in the immunocytochemical test, showing a decrease in the distribution of protein membrane in the mutant channel of A422T and H562P, the increase in the cytoplasmic protein, and the co loci with the endoplasmic reticulum, indicating that the two mutant channel proteins are due to the transport barrier. It was stuck in the endoplasmic reticulum.
2. secondly, the plasmids of WT and A422T or H562P were converted to HEK 293 cells. The results of whole cell patch clamp technique and Western blot detection showed that the current and mature protein expression of the heterozygous channel were significantly less than that of WT, and the channel power and protein expression were aggravated with the increase of the particle quantity of the transfection mutation, indicating A422T and H562P to WT. The current and protein expression has a concentration dependent negative dominance inhibition effect. Further analysis of the electrophysiological characteristics of A422T/WT (transfected plasmid 1:1, simulated patient's common heterozygous genotype), H562P/WT (transfected plasmids as 1:1, simulated patient common heterozygous genotypes), the results showed that the expression of the mature protein was expressed. In addition, the kinetics of heterozygous channel changes compared with that of WT, and A422T/WT is mainly manifested in the slow activation process and the H562P/WT performance is accelerated in the inactivation process. Compared with A422T/WT, H562P/WT has the characteristics of faster activation and faster inactivation, which will shorten the opening time of the channel, and thus significantly affect the potassium ionization of the action potential repolarization. This change in kinetics is another reason for the dysfunction of heterozygous channels.
3. further use different methods to treat A422T, H562P mutation channel, compare the salvage effect of the current and protein expression, analyze the structure of the two mutant and the molecular mechanism of the transport barrier. Under the condition of low temperature or E4031 treatment, the function of the A422T mutation channel and the transfer of protein can be significantly improved, not only to prove A422T again, but not only again. The mutant channel protein transport barrier occurs in the endoplasmic reticulum and indicates that the mutant channel protein can form the basic four polymer structure and pore structure, which is a wrong folding and not serious mutation protein. On the contrary, the two treatments are invalid for the H562P mutation channel, indicating that the protein folding error is serious and can not be saved by the common methods.
In the case of overexpression of Hsp70 and / or Hsp90, Co-IP detection confirmed the interaction between A422T or H562P and Hsp70 and Hsp90 three, and the binding amount of the mutant channel protein to Hsp70 or Hsp90 is different compared with the wild type HERG potassium channel protein, and the overexpression of Hsp70 and Hsp90 has partially saved the current of the mutation channel. There is no significant effect of protein expression; this salvage effect exists in the condition of single overexpression of Hsp90 rather than Hsp70. The inhibitor of tripterin - Hsp90 cancels its salvage effect. These indicate that Hsp70 and Hsp90 are all involved in the transport process of HERG potassium channel protein, but Hsp90 is more important for the protein transport of A422T. In spite of the interaction with Hsp70 and Hsp90, the expression of Hsp70 and / or Hsp90 has no significant effect on its function and protein expression, further explaining the missense mutations in two different locations, and the mechanism that affects its function can not be corrected by the quality control system of the endoplasmic reticulum.
Under the moderate hypoxia stimulation (2%O2 action 24hr), it has a significant effect on the current and protein maturation of the WT channel, but also can restore the functional part of the A422T mutation channel, but has no effect on H562P. It suggests that appropriate endoplasmic reticulum stress (ER stress) can promote the transport and maturation of the egg white in the potassium channel of HERG.
4. finally, using the reverse transcriptional PCR test technique to detect the shear condition of XBP1, to understand whether the two mutants are overstacked in the cells and cause the ER stress. experimental results to show that the A422T and H562P mutant channel proteins are blocked in the endoplasmic reticulum, but do not accumulate too much and do not cause ER stress, but enter the ubiquitin proteasome pathway in time. Degrading.
Conclusion:
1.HERG missense mutations A422T and H562P cause the barrier of channel protein transport and block in the endoplasmic reticulum, which is a cellular mechanism that causes loss of channel function and causes LQTS (LQT2).
2. The A422T mutant is a non-misfolded protein that can form a tetramer structure and restore some of its functions at low temperatures, drugs, molecular chaperones or hypoxia.
The 3.H562P mutant has no effective method to save its function. It may be because H562P is located in the S5 transmembrane fragment, located inside the channel, and the position is key and concealed, and it is difficult to correct.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 劉文玲,胡大一,李翠蘭,李萍,李運(yùn)田,李志明,李蕾,秦緒光,董瑋,戚豫,陳勝寒,王擎;Mutation analysis of potassium channel genes KCNQ1 and KCNH2 in patients with long QT syndrome[J];Chinese Medical Journal;2003年09期
，本文編號(hào)：2170282
本文鏈接：http://sikaile.net/xiyixuelunwen/2170282.html
最近更新
教材專著
