长QT综合征相关基因新突变G52R-KCNE1的功能研究

目的 了解长QT综合征相关基因新突变G52R-KCNE1的功能。方法 采用交叠PCR方法体外制备突变体G52R-KCNE1,并克隆到原核细胞表达载体中;体外合成RNA、显微注射入卵母细胞,在卵母细胞中共表达野生型KCNQ1(WT-KCNQ1)、野生型KCNE1(WT-KCNE1)和突变体G52R-KCNE1,采用标准双电极电压钳了解突变体的功能。结果 突变体G52R-KCNE1不能放大WT-KCNQ1通道的电流强度;当WT-KCNE1和G52R-KCNE1等量注射时,G52R-KCNE1可降低WT-KCNQ1／WT-KCNE1通道约50％的电流强度,而不影响该通道的激活动力学。结论 位于跨膜区第52位的甘氨酸对维持KCNE1的功能非常重要;突变体G52R-KCNE1明显抑制WT-KCNE1通道的功能,这可能是突变基因携带者心肌细胞复极化延缓,QT间期延长的分子电生理机制。     

QT延长综合征HERG基因新突变位点E505D的功能检测

目的 研究中国人QT延长综合征2型(LQT2)HERG基因突变(E505D)导致的功能改变。方法 将HERG基因野生型cRNA和突变型(E505D)cRNA分别和同时注射到非洲爪蟾卵母细胞,采用双电极电压钳技术记录非洲爪蟾卵母细胞膜上电流的表达,了解基因突变对电流的影响。结果 单独注射突变型cRNA记录到的电流与注射双蒸水记录到的电流差异无显著性。野生型和突变型cRNA共同注射记录到的电流显著低于单独注射野生型cRNA,电流改变呈负电位占优势的方式。结论 HERG基因突变使快速激活延迟整流性钾通道电流减小,心室复极时间延长,QTc延长,证实HERG基因突变与LQT2相关。     

HERG错义突变V630A和N633S引起QT延长综合征的分子机制

目的 HERG基因突变可引起遗传性QT延长综合征（10ng QT syndroms,LQTS）。研究探讨HERG通道孔区的错义突变导致LQTS的分子机制。方法 采用Megapfimer方法制备HERGV630A、N633S突变体,并亚克隆到pSP64和pcDNA3．1表达载体。用T7RNA聚合酶体外合成cRNA,经Ribogreen荧光定量后,将相应的cRNA注入卵母细胞,在18℃培养箱中培养3d后,用标准双电极电压钳技术测量卵母细胞的表达电流。所有数据均用pCLAMP软件采集,并应用Kaleidagraph 3．5和Igor软件进行数据处理。结果 当V630A、N633S分别同HERG野生型在卵母细胞共同表达时,这两种突变体均具有明显的负显性抑制效应,其抑制效应达到50％～70％。同野生型HERG通道相比,这两种异源多聚体的失活显著加快,半失活电压负向偏移。结论 该研究首次阐明了两个位于HERG通道孔区外口的错义突变V630A、N633S所引起的通道电生理学功能改变,从分子水平证明HERG通道孔区的错义突变与致命性心律失常的关系。     

交感神经兴奋与1型长QT综合征(LQT1)

遗传性长QT综合征（LQTS）是一种严重危害人类健康的心律失常,其中1型长QT综合症（long QT syndrome type 1,LQT1）是由KCNQ1基因突变所致。KCNQ1基因编码心脏缓慢激活延迟性整流钾电流（I_Ks）,离子通道（Kv7.1）的α亚基。KCNQ1基因发生突变,可以造成心脏复极主要外向电流之一的I_Ks通道功能受损,结果导致心肌细胞动作电位时程（APD）和心电图QT间隔延长。研究结果表明,LQT1患者发生的心脏意外事件通常与交感神经兴奋有关（例如运动或情绪激动）,特别是游泳和潜水。本文就交感神经兴奋与LQT1的关系进行阐述,并对相关的预防、诊断与治疗措施进行总结。     

老年大鼠耳蜗核钾离子通道在爪蟾卵母细胞中的表达

目的观察爪蟾卵母细胞表达的老年大鼠耳蜗核电压依赖性离子通道电流的变化，为研究感音神经性聋听觉中枢离子通道的变化建立方法学基础。方法分别自青年和老年大鼠耳蜗核提取多聚腺嘌呤信使核糖核酸（ｍＲＮＡ），注入非洲爪蟾卵母细胞表达有功能的钾离子通道，并利用电压钳方法记录电压依赖性的离子通道电流。结果在注射老年大鼠耳蜗核ｍＲＮＡ的卵母细胞膜上记录到电压依赖性的钾离子通道电流最大幅度（２７８±３７ｎＡ，１６只），明显小于青年组（３６４±４２ｎＡ，１７只，Ｐ＜０００１）。结论移植的老年大鼠耳蜗核钾离子通道功能的下降可能与老年性聋的发病有关     

先天性长QT综合征KCNQ1基因定点突变的研究

目的利用聚合酶链反应(PCR)技术对长QT综合征(LQTS)KCNQ1基因进行定点突变的研究。方法利用PCR定点突变技术，首先设计两对引物(包含预定的突变)，通过3轮PCR扩增，扩增出含有所需突变位点的片段，然后将片段克隆入T载体中，通过酶切连接的方法将突变点引入到pIRES2-EGFP-KCNQ1中，随后用Effectene转染试剂介导转染HEK293细胞。结果在真核表达载体pIRES2-EGFP—KCNQ1基础上获得了KCNQ1 cDNA C682T的突变体，测序结果表明在序列中发生了预期的突变。结论KCNQ1定点突变体的构建为进一步的功能研究奠定了基础。     

先天性QT延长综合征亚型JLN综合征的基因突变研究

目的　对一先天性QT延长综合征亚型JLN综合征 (JLNS)家系进行基因突变检测 ,以期发现中国人特有的JLNS突变。方法　采用聚合酶链反应及直接测序法对一JLNS家系进行KCNQ1及KCNE1的基因检测。结果　在先证者及其姐姐的KCNQ1基因的第 1 5外显子发现一错义突变 :2 0 37(G→A)、G6 4 3S ,还有另一突变是KCNQ1基因外显子 2a的第 2 2 7位核苷酸C被T代替 ,其编码的苏氨酸被异亮氨酸所代替。而这两突变分别来自其表型正常的父母亲。结论　JLNS可由KCNQ1基因上复合的杂合突变所引起。在中国QT延长综合征患者中发现两个JLNS的新的突变。     

用于离子通道基因表达的爪蟾卵母细胞的分离和培养

应用非洲爪蟾卵母细胞对外源性离子通道基因进行表达是目前离子通道结构和功能研究中的一种重要的方法 ,它实现了分子生物学和电生理实验的很好结合。作者基于离子通道研究的实验需要 ,提出了一种用于离子通道基因表达的卵母细胞的制备方法。     

先天性长QT综合征KVLQT1和HERG基因新突变位点的检测

目的：研究中国人先天性长QT综合征（long QT syndrome,LQTS)HERG和KVLQT1的基因突变情况。方法,利用聚合酶链反应和DNA测序对11个LQTS家系HERG跨膜编码区S1－S6和KVLQT1跨膜编码区S2－S6进行基因检测。结果（1）11个LQTS患者在国外已知突变点均无突变;（2）发现4个新的错义突变位点,分别为T1515G（HERG）,C682T,C934T,G983A（KVLQT1）。其对应的氨基酸改变为E505D,R228C,S230L,P312S和R328C。结论：在中国人LQTS患者HERG和KVLQT1上发现了4个新的基因突变位点。     

利用爪蟾卵母细胞建立人类低密度脂蛋白受体基因表达系统

目的在爪蟾卵母细胞中建立人类低密度脂蛋白受体基因的表达系统,此系统可被用于降血脂和抗动脉粥样硬化的研究.方法和结果冰块麻醉爪蟾,切取卵巢,获得Ⅴ、Ⅵ期卵母细胞,经用1g/L胶原酶孵育过夜除去卵母细胞外层的卵巢膜和滤泡膜,培养3天后存活率仍可达95%以上.用DiI标记的低密度脂蛋白荧光配体测定膜上结合荧光,发现Ⅴ、Ⅵ期卵母细胞膜上未见有内源性低密度脂蛋白受体,如将人低密度脂蛋白受体的表达质粒p3.7低密度脂蛋白导入爪蟾卵母细胞核中,培养后用DiI标记的低密度脂蛋白配体测定可见膜上发出红色荧光,用抗低密度脂蛋白受体的抗体经免疫荧光检测法测定可见膜上有绿色荧光,免疫胶体金标记透射电镜检测可见膜上有内吞的胶体金颗粒.结论外源性的人类低密度脂蛋白受体基因能在爪蟾Ⅴ、Ⅵ期卵母细胞中表达,此低密度脂蛋白受体基因表达系统可用于中药降血脂抗动脉粥样硬化机理的研究.     

普罗帕酮对HERG钾通道孔胞膜外侧氨基酸突变前后的作用

目的:探讨普罗帕酮对人类ether-a-go-go相关基因(HERG)钾通道孔道胞膜外侧突变后结合能力的影响。方法:将HERG野生型通道(WT)和HERG突变型通道(MT)的互补核糖核酸(cRNA)注射到非洲爪蟾卵母细胞,孵育24～72h后以不同刺激程序用双微电极法记录通道电流的表达。用Clampfit9.2版本软件对数据进行分析。部分电流用相应的方程拟合。结果:HERGWT外侧的第628位点的甘氨酸突变为半胱氨酸(G628C)和第631位点的丝氨酸突变为半胱氨酸(S631C)成HERGMT后普罗帕酮与HERGMT通道的结合能力减小,50%抑制浓度(IC50)对HERGWT,HERGMT分别为5.31μmol/L和7.81μmol/L。普罗帕酮对HERGWT和HERGMT的阻滞效应都呈电压和浓度依赖性。普罗帕酮减小HERGWT,但未减少HERGMT通道的半数激活电压。结论:普罗帕酮是HERG通道的开放通道阻滞剂,HERG通道孔道膜外侧突变(G628C和S631C)能改变普罗帕酮与通道之间的结合能力,从而影响通道的激活。     

Characterization of a novel Long QT syndrome mutation G52R-KCNE1 in a Chinese family

OBJECTIVES: To identify the underlying genetic basis of a Chinese pedigree with Long QT syndrome, the causally related genes were screened in a family and the functional consequence of the identified gene mutation was evaluated in vitro. METHODS: Mutations in the five defined Long QT syndrome related genes were screened with polymerase chain reaction and single-strand conformation polymorphism methods and direct sequencing. The electrophysiological properties of the identified mutation were characterized in the Xenopus oocyte heterologous expression system. RESULTS: A novel missense mutation, G to A at position 154 in the KCNE1 gene was identified in a Chinese Long QT syndrome family, which leads to an amino acid substitution of arginine (R) for glycine (G) at position 52 (G52R-KCNE1). Of 26 family members (one DNA was not available), seven were mutation carriers and two of them with normal electrocardiogram. Compared with wild-type KCNE1/KCNQ1 channels, coexpression of G52R-KCNE1 with KCNQ1 in Xenopus oocytes did not amplify the KCNQ1 current amplitudes and slightly changed the activation kinetics of the KCNQ1 channels. Coexpression of KCNQ1 together with wild type KCNE1 and G52R-KCNE1 reduced the wild-type I(ks) current amplitude by 50%, whereas other biophysical properties of the I(ks) were not altered. CONCLUSIONS: Our findings indicate that glycine52 in the transmembrane domain is critical for KCNE1 function. The mutant G52R-KCNE1 has a dominant negative effect on I(ks) current, which reduces the I(ks) current amplitude and leads to a prolongation of the cardiac action potential. This could underlie the molecular mechanism of ventricular arrhythmias and sudden death in those patients.     

KCNQ1 mutation Q147R is associated with atrial fibrillation and prolonged QT interval

BACKGROUND: Atrial fibrillation (AF) and long QT syndrome (LQTS) are cardiac arrhythmia disorders that have been related to dysfunction of the voltage-gated potassium channel subunit Kv7.1 encoded by the KCNQ1 gene. OBJECTIVE: The purpose of this study was functional assessment of a mutation in Kv7.1 identified in a proband with permanent AF and prolonged QT interval. We investigated whether this KCNQ1 missense mutation could form the genetic basis for AF and LQTS simultaneously in this patient. METHODS: We investigated the functional consequences of the novel mutation KCNQ1 Q147R by heterologous expression of the channel and accessory subunits in Xenopus laevis oocytes and mammalian cells. RESULTS: The Q147R mutation does not affect the biophysical properties of Kv7.1 in the absence of accessory subunits. Upon coexpression with the beta-subunit KCNE1, the Q147R mutation induced a loss of function, observed as a decrease in current amplitude at depolarized potentials. Additionally, Q147R abolished the frequency dependence of charge carried by Kv7.1/KCNE1 channels. Coexpression with the beta-subunit KCNE2 revealed a gain of function for the mutant, seen as an increase in the current amplitude at depolarized potentials. The properties of channels formed by Kv7.1 and the subunits KCNE3 and KCNE4 were unaffected by the Q147R mutation. CONCLUSION: Our data indicate that the Q147R mutation can form the molecular substrate simultaneously for different arrhythmogenic conditions. The mechanism may be heterogeneous distribution of Kv7.1 accessory subunits in the heart leading to Kv7.1 gain of function in the atria (for AF) and Kv7.1 loss of function in the ventricles (for QT prolongation).     

A novel LQT3 mutation implicates the human cardiac sodium channel domain IVS6 in inactivation kinetics

The Long QT3 syndrome is associated with mutations in the cardiac sodium channel gene SCN5A. OBJECTIVE: The aim of the present study was the identification and functional characterization of a mutation in a family with the long QT3 syndrome. METHODS: The human cardiac sodium channel gene SCN5A was screened for mutations by single-stranded conformation polymorphism. The functional consequences of mutant sodium channels were characterized after expressing mutant and wild-type cRNAs in Xenopus oocytes by two-electrode voltage clamp measurements. RESULTS: SCN5A screening revealed an A-->G substitution at codon 1768, close to the C-terminal end of domain IVS6, which changes an isoleucine to a valine. Functional expression of mutant I1768V-channels in Xenopus oocytes showed that the voltage-dependence and slope factors of activation and inactivation were unchanged compared to wild-type channels. No difference in persistent TTX-sensitive current could be detected between wild-type and I1768V channels, a channel feature often increased in LQT3 mutants. However, I1768V mutant channels recovered faster from inactivation (2.4 times) than wild-type channels and displayed less slow inactivation. CONCLUSIONS: We postulate that severe destabilization of the inactivated state leads to increased arrhythmogenesis and QT prolongation in I1768V mutation carriers in the absence of a persistent inward sodium current.     

Liddle's syndrome caused by a novel missense mutation (P617L) of the epithelial sodium channel beta subunit

OBJECTIVE: The aim of the study was to search for mutations of SCNN1B and SCNN1G in an Italian family with apparently dominant autosomal transmission of a clinical phenotype consistent with Liddle's syndrome. METHODS: Genetic analysis was performed in the proband, his relatives, and 100 control subjects. To determine the functional role of the mutation identified in the proband, we expressed the mutant or wild-type epithelial sodium channel in Xenopus laevis oocytes. RESULTS: A novel point mutation, causing an expected substitution of a leucine residue for the second proline residue of the conserved PY motif (PPP x Y) of the beta subunit was identified in the proband. The functional expression of the mutant epithelial sodium channel in X. laevis oocytes showed a three-fold increase in the amiloride-sensitive current as compared with that of the wild-type channel. CONCLUSION: This newly identified mutation adds to other missense mutations of the PY motif of the beta subunit of the epithelial sodium channel, thus confirming its crucial role in the regulation of the epithelial sodium channel. To our knowledge, this is the first report of Liddle's syndrome in the Italian population, confirmed by genetic and functional analysis, with the identification of a gain-of-function mutation not previously reported.     

普罗帕酮对Kv1.4通道内口突变前后的影响

目的探讨普罗帕酮对编码瞬时外向钾电流(Ito)慢通道Kv1.4通道(fKv1.4ΔN)内口突变(fKv1.4[V561A]ΔN)前后的影响。方法将fKv1.4ΔN和fKv1.4[V561A]ΔN的cRNA注射到非洲爪蟾卵母细胞,孵育24～72 h后使用不同刺激程序用双微电极法记录通道电流的表达。用Clampfit 9.0对数据进行分析。部分电流用相应的方程拟合。结果普罗帕酮对fKv1.4ΔN和fKv1.4[V561A]ΔN的阻滞效应都呈电压和频率依赖性。通道内口的V561A突变使fKv1.4ΔN通道与普罗帕酮的结合能力减小,50%抑制浓度(IC50)在突变前后分别为100μmol/L和380μmol/L(P<0.01)。普罗帕酮能改变fKv1.4ΔN和fKv1.4[V561A]ΔN的失活特性。尽管普罗帕酮对fKv1.4ΔN的失活后恢复没有影响,但是它能延长fKv1.4[V561A]ΔN的50%失活后恢复时间。结论普罗帕酮是fKv1.4ΔN通道的开放通道阻滞剂,fKv1.4ΔN通道内口突变(V561A)能改变普罗帕酮与通道之间的结合能力,从而影响通道的失活和失活后恢复。