KCNQ1通道突变致病机制及乙醇作用的研究

发布时间：2018-04-23 00:32

本文选题：离子通道 + KCNQ家族　；参考：《华中科技大学》2009年博士论文

【摘要】： 离子通道是一种能够调节细胞膜两侧离子流的融合蛋白，它是神经、肌肉和其它组织细胞膜兴奋的基础，也是生物电活动的基础。钾离子通道是迄今为止类型最多的一类离子通道，它们广泛地分布于骨骼肌、神经、心脏、血管、气管、胃肠道、血液、内分泌和腺体等细胞。KCNQ基因编码的钾离子通道家族是电压门控钾离子通道的一个重要分支，在心脏中大量表达，在内耳也有分布。KCNQ1及其辅助亚基minK形成的KCNQ1／minK复合体能够产生延迟外向整流钾电流I_(KS)，该电流帮助终结心肌细胞中的动作电位，KCNQ1基因若发生突变，就会引起该通道的功能紊乱，从而引起心脏长QT综合征，即LQTS，最终导致严重的心律不齐，室颤甚至心脏休克。目前发现许多疾病与KCNQ基因突变或其编码的钾离子通道功能失调有关，鉴于这些原因，弄清楚KCNQ1通道的相关特性，无论对于离子通道学，还是临床医学都会具有很大的贡献。本文在HEK293细胞上研究了KCNQ1突变基因L191P导致LQT1的机制。在KCNQ1基因中有超过100种突变是引发1型LQTS(LQT1)的主要原因，有报道发现，位于1 91位点的亮氨酸突变L1 91 P能够直接引发LQT1，该突变点位于KCNQ1基因的S2-S3连接区域，大约有16％的LQTS突变点位于该区域中。在电生理实验结果中，我们发现L1 91 P／minK产生的电流远远小于正常的WT／minK的电流，电流-电压(IV)曲线中显示L191 P／minK的电流减少到WT／minK的电流的一半以下，但是电导-电压(GV)曲线中显示没有明显的变化。在免疫荧光成像结果中，我们却发现L191 P使得正常的KCNQ1蛋白上膜量大大降低，因此推断该突变通道因上膜量剧烈变化使得心肌钾电流I_(Ks)大大减小，从而影响动作电位复极化，导致LQTS的产生。为了更进一步了解这种上膜量的变化原因，我们将这个点的氨基酸残基Leu突变成疏水性不同的氨基酸(Phe＞Leu＞Val＞Trp＞Ala＞Pro＞Lys＞Asp)，发现这些突变随着其疏水性的减弱，其上膜能力也逐渐减弱。通过建立E(?)M模型，我们归纳出这一规律遵循的波尔兹曼公式，应用于因能量变化而改变膜蛋白上膜效率的情况。同时，通过建立二项式分布模型，我们解释了dominant-negative效应的存在是LQT1表型存在的本质。本文还在非洲爪蟾Xenopus laevis卵母细胞上研究了乙醇(酒精)阻断KCNQ1通道的机制。乙醇对人体具有广泛的药理学影响，研究者们知道乙醇对脑、心脏和肝脏等的功能会产生不良影响，但不了解它起作用的机制。我们通过双电极电压钳检测，结果表明乙醇能够特异性阻断I_(Ks)电流，与其类似的其它直链烷醇亦能够阻断该通道，而且烷醇链长越长，相同浓度下阻断能力越强。这种阻断效果不仅具有电压依赖性，还同时兼具关闭态阻断和开放态阻断的特点，说明酒精阻断KCNQ1通道的作用位点可能既存在于细胞外，也存在于细胞内。通过突变扫描比对，我们还发现KCNQ1上氨基酸Ile257在酒精等烷醇对KCNQ1通道的阻断过程中起着重要的作用。我们还借助MedLab生物信号采集处理系统，连续测量注射不同浓度乙醇的小鼠心电图，结果表明一定浓度下乙醇(较低或者很高浓度)能够阻断心肌钾离子通道，从而延迟动作电位的复极化，，进而延缓心率；而某一特定浓度下的乙醇(较高安全浓度)能够刺激心肌钾离子通道的开放，从而加速动作电位的复极化，进而加速心率。我们利用了“口袋”模型进一步阐明烷醇和KCNQ1通道相互作用机制，这对以KCNQ1通道为靶点的药物研究有着重要意义。
[Abstract]:Ion channel is a fusion protein that regulates the ion flow on both sides of the cell membrane. It is the basis of the excitement of nerve, muscle and other tissue cell membranes. It is also the basis of bioelectrical activity. Potassium channels are the most types of ion channels so far. They are widely distributed in skeletal muscles, nerves, heart, blood vessels, trachea, and gastrointestinal tract. The.KCNQ gene coded potassium channel family of blood, endocrine and glandular cells is an important branch of the voltage-gated potassium channel, which is widely expressed in the heart, and the KCNQ1 / minK complex, formed by the distribution of.KCNQ1 and its auxiliary subunit minK in the inner ear, can produce a delayed extroverted rectifier potassium current I_ (KS), which helps to end the current. The action potential in the cardiac myocytes, if the KCNQ1 gene mutation, causes the dysfunction of the channel, causing the heart long QT syndrome, that is, LQTS, which eventually leads to severe arrhythmia, ventricular fibrillation and even cardiac shock. Many diseases are currently found to be associated with the KCNQ gene mutation or its coded potassium channel dysfunction, given these The reasons for identifying the related properties of KCNQ1 channels will contribute greatly to both ion channels and clinical medicine.
In this paper, the mechanism of the KCNQ1 mutation L191P causing LQT1 was studied on HEK293 cells. More than 100 mutations in the KCNQ1 gene are the main causes of the type 1 LQTS (LQT1). It is reported that the leucine mutation at the 191 site, L1 91 P, can directly trigger LQT1, which is located in the S2-S3 junction region of the KCNQ1 gene, about 16% In the electrophysiological experiment, we found that the current produced by L1 91 P / minK is far less than the normal WT / minK current, and the current voltage (IV) curve shows that the current of L191 P / minK decreases to less than half of the current of WT / minK, but there is no obvious change in the conductance voltage curve. In the results of immunofluorescence, we found that L191 P greatly reduced the amount of the normal KCNQ1 protein on the membrane. Therefore, it was inferred that the abrupt change in the amount of the upper membrane caused the myocardial potassium current I_ (Ks) to be greatly reduced, thus affecting the repolarization of the action potential and leading to the production of LQTS.
In order to further understand the cause of the change in the amount of the membrane, we turn the amino acid residue Leu of this point into a hydrophobic amino acid (Phe > Leu > Val > Trp > Ala > Pro > Lys > Asp). It is found that these mutations decrease with their hydrophobicity weakening. By establishing E (?) M model, we conclude this A regular Boltzmann formula is applied to change the membrane efficiency of membrane proteins due to energy changes. At the same time, by establishing a binomial distribution model, we explain that the existence of the dominant-negative effect is the essence of the existence of the LQT1 phenotype.
This paper also studies the mechanism of ethanol (alcohol) blocking the KCNQ1 channel on the Xenopus laevis oocytes of Xenopus Xenopus. Ethanol has extensive pharmacological effects on human body. Researchers know that ethanol has adverse effects on brain, heart, liver and other functions, but does not understand the mechanism of its effect. We use dual electrode voltage tweezers to detect the effects of ethanol. The results show that ethanol can specifically block the I_ (Ks) current, and its similar direct chain alkanols can block the channel, and the longer the alkanol chain length, the stronger the blocking ability under the same concentration. This blocking effect not only has voltage dependence, but also has the characteristics of closed state blocking and open state blocking, indicating that alcohol is blocking KCNQ1. The site of the channel may exist both in the cell and in the cell. Through the mutation scan comparison, we also found that the amino acid Ile257 on KCNQ1 plays an important role in the blocking of KCNQ1 channels.
We also use the MedLab biological signal acquisition and processing system to continuously measure the electrocardiogram of mice injected with different concentrations of ethanol. The results show that ethanol (lower or very high concentration) at a certain concentration can block the potassium channels of the myocardium, thus delaying the repolarization of the action potential and postponing the heart rate; and the higher alcohol (higher) at a certain concentration (higher) Safety concentration stimulates the opening of the potassium channels of the myocardium, thus accelerating the repolarization of the action potential and accelerating the heart rate. We use the "pocket" model to further elucidate the interaction mechanism of alkanols and KCNQ1 channels, which is of great significance for the study of drugs targeting the KCNQ1 channel.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363.2

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