【作者】 潘娜；

【导师】 丁久平；

【作者基本信息】 华中科技大学 ， 生物物理学， 2009， 博士

【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。更多还原

【Abstract】 Ion channel, a fusion protein regulating transmembrane ion flux, is the basis of bio-electrical activity and the membrane excitablity of nerve, muscle and other tissu. Potassium channels are the most various ion channels, and widely exist in skeletal muscle, nerve, heart, vessels, trachea, gastrointestinal tract, blood and endocrine gland cells. KCNQ1 channel, which is an important branch of voltage-gated potassium channels, has a large distribution in the heart, as well as the inner ear. Combined with its auxiliary subunit minK, KCNQ1/minK complex can generate a delayed outward rectifier K⁺ current, Iks. to end the action potential in myocardial cells, therefore, KCNQ1 mutations would cause a dysfunction of the channel, thus give rise to the long QT syndrome （LQTS）, eventually lead to serious arrhythmias, ventricular fibrillation and cardiac shock. Mutations in KCNQ gene can lead to many diseases, thus, it is very contributing to clarify the characteristics of KCNQ1 channels, both for the ion channel research and clinical medicine.We studied the mechanism of LQT1 arising from one KCNQ1 mutation, L191P, in HEK293 cells. Over 100 KCNQ1 mutantions can lead to the type-1 LQTS （LQT1）. It is reported that KCNQ1-Leu191, which locates in the intracellular S2-S3 linker of KCNQ1, can lead to LQT1 directly, and～16% LQTS-related mutations mustered in this region. By electrophysiological method, we found the currents of L191P/minK channel were much smaller than those of KCNQ1/minK channels. Although the average currents of L191P/minK were reduced to more than half compared with those of WT/minK, but there was no shift in the G-V curves. By immunofluorescence method, we also found L191P channels causing a trafficking deficiency, leading to the minishing of I_Ks and then, LQTS.We found the surface expression decreased with decreasing hydrophobicity of the middle residue ’X’ of the RXR motif. Establishing a model of E（?）M, we generalize the Boltzmann formula to explain the results, and we got the essence of LQT1 phenotype through the binomial distribution model, that is the dominant-negative effect.We also studied the blocking mechanism of ethanol （alcohol） on KCNQ1 in Xenopus laevis oocytes. Ethanol has a wide range of pharmacological effects on the human body, but researchers do not understand the mechanism it works. Using two-electrode voltage clamp, we showed that ethanol blocked specificly I_Ks, even the straight-chain n-alcohol also blocked the channel. The chain length was longer, the same concentration blocked stronger. The voltage-dependent and bio-states blocking showed us that ethanol blocked the channel both from outside and inside. Through mutation scanning, we also found that amino acid Ile257 of KCNQ1 played an important role in the blocking.We also used the Medlab bio-signal acquisition and processing system to measure the ECG of the injected mice, and results showed that a certain concentration of ethanol blocked cardiac potassium channels, which delayed action potentials repolarization and thus slowed down the heart rate; and a particular concentration of ethanol （higher over security levels） stimulated the cardiac potassium ion channels opening, thus speeded up the action potential repolarization, thus speeded up the heart rate. We used a "pocket" model to further clarify the interaction beteween n-ethanol and KCNQ1 channel, which will make a graet sense to the study of the pathogenic mechanisms related to heart disease.更多还原