HERG错义突变A422T和H562P导致LQT综合征的细胞分子机制
发布时间：2018-08-07 14:13
【摘要】：HERG钾通道电流是心肌细胞动作电位的重要组分,在3期复极化晚期发挥关键作用。药物的阻断作用或HERG基因突变造成该电流减少,可分别导致获得性LQTS和遗传性LQTS (LQT2)。就LQT2而言,目前已发现的突变位点有200多个,但致病机制各不相同。 A422T和H562P是从两个不同家庭LQTS病人筛选到的错义突变位点,电生理分析结果显示,这两种不同位点突变通道电流明显减小,功能缺失,但具体的发生机制是何,尚不清楚。本课题利用膜片钳技术结合细胞分子生物学技术,研究分析A422T和H562P突变导致HERG钾通道功能缺失的机制,并探讨不同方法的挽救效果,为理解通道蛋白运输缺陷导致HERG钾通道功能缺失、造成LQT2的细胞分子机制提供详实的实验依据,同时为LQTS疾病的临床治疗提供新视点。 主要结果如下： 1.首先,利用全细胞膜片钳技术,对分别表达A422T和H562P突变通道的HEK 293细胞进行检测,结果发现两种错义突变均可导致HERG钾通道电流幅度减少,电压依赖性消失,说明A422T和H562P存在不同程度的功能缺失。Western blot实验结果显示,A422T和H562P通道的155 kDa成熟蛋白组分消失,提示突变蛋白存在转运障碍。这一结果进一步在免疫细胞化学检测实验得到证实,表现在A422T与H562P突变通道蛋白膜分布减少,胞质内蛋白增加,且与内质网共定位,说明两种突变通道蛋白由于转运障碍而被滞留在内质网内。 2.其次,将WT与A422T或H562P的质粒共转HEK 293细胞,利用全细胞膜片钳技术及western blot检测的结果显示,杂合型通道的电流及成熟蛋白表达均显著少于WT,并且随着转染突变体质粒量的增加,通道功能和蛋白表达受损加重,说明A422T、H562P对WT的电流及蛋白表达有浓度依赖的负显性抑制作用。进一步对A422T/WT(转染时质粒的量为1：1,模拟病人常见杂合基因型)、H562P/WT(转染时质粒的量为1：1,模拟病人常见杂合基因型)的电生理特性进行检测分析,其结果表明,除成熟蛋白表达减少外,杂合型通道的动力学与WT相比也有改变,A422T/WT主要表现在激活过程减慢,而H562P/WT表现在失活过程加快。而与A422T/WT相比,H562P/WT具有更快激活,更快失活的特点,这将使通道得开放时间缩短,进而显著影响动作电位复极化的钾离子流。这种动力学的改变是导致杂合型通道功能缺陷的另一原因。 3.进一步利用不同方法对A422T、H562P突变通道进行处理,比较其对电流及蛋白表达的挽救作用,分析两突变体的结构及转运障碍的分子机制。在低温或E4031处理条件下,A422T突变通道的功能和蛋白转运均可得到显著改善,不但再次证明A422T突变通道蛋白转运障碍发生于内质网,并且说明该突变通道蛋白可形成基本的四聚体结构及孔道结构,系一种错误折叠不严重的突变蛋白；相反,两种处理对H562P突变通道却无效,说明其蛋白折叠错误严重,无法被常用方法挽救。 在过表达Hsp70和/或Hsp90的情况下,Co-IP检测证实A422T或H562P与Hsp70和Hsp90三者之间存在相互作用,并且与野生型HERG钾通道蛋白相比,突变通道蛋白与Hsp70或Hsp90的结合量也不同；过表达Hsp70和Hsp90对A422T突变通道电流有部分挽救作用,而对蛋白表达没有显著影响；这种挽救作用存在于单独过表达Hsp90而不是Hsp70的条件下,雷公藤红素——Hsp90的抑制剂可取消其挽救作用。这些说明Hsp70、Hsp90均参与HERG钾通道蛋白的转运过程,但Hsp90对A422T的蛋白转运更为重要。对H562P突变通道而言,尽管与Hsp70和Hsp90存在相互作用,但无论过表达Hsp70和/或Hsp90,对其功能和蛋白表达都无明显影响,进一步说明两种不同位置的错义突变,影响其功能的机制存在差异。H562P突变无法被内质网质量控制系统纠正。 在适度低氧刺激(2%O2作用24hr)下,对WT通道电流及蛋白成熟过程均有明显促进作用,也可使A422T突变通道功能部分恢复,但对H562P无影响。提示适当的内质网应激(ER stress)可以促进HERG钾通道蛋白的转运、成熟。 4.最后,利用反转录PCR实验技术检测XBP1的剪切情况,了解两突变体是否在细胞内过度堆积,引起ER stress。实验结果表明A422T和H562P突变通道蛋白虽然阻滞于内质网中,但并未过多堆积,也未造成ER stress,而是及时进入泛素-蛋白酶体途径进行降解。 结论： 1.HERG错义突变A422T和H562P使通道蛋白转运障碍并阻滞于内质网,是导致通道功能缺失并引起LQTS (LQT2)的细胞分子机制； 2.A422T突变体是错误折叠不严重的蛋白,可形成四聚体结构,在低温、药物、分子伴侣或低氧处理后可恢复部分功能； 3.H562P突变体,尚没有有效的方法挽救其功能。可能是由于H562P位于S5跨膜片段,位于孔道内部,位置关键且隐蔽,难以纠正。
[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.
【学位授予单位】：南京师范大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
本文编号：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.
【学位授予单位】：南京师范大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
【参考文献】
相关期刊论文 前1条
1 刘文玲,胡大一,李翠兰,李萍,李运田,李志明,李蕾,秦绪光,董玮,戚豫,陈胜寒,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
，本文编号：2170282
本文链接：https://www.wllwen.com/xiyixuelunwen/2170282.html
最近更新
教材专著
