【作者】 韩圣娜；

【导师】 张莉蓉；

【作者基本信息】 郑州大学 ， 药物不良反应， 2010， 博士

【摘要】 长QT综合症(long QT syndrome,LQTS)可分为先天遗传性和后天获得性LQTS二类。先天性LQTS是由于编码心肌细胞离子通道的基因突变导致相应离子通道蛋白功能发生异常所致。目前已知的先天性LQTS的相关基因至少有十种,分别为LQT1-LQT10。其中LQT2是由于编码快速激活的延迟整流钾通道(rapid activating delayed rectifier K+ channel,IKr)的hERG基因(human ether-a-go-go-related gene,hERG)发生突变所致,IKr通道是形成心肌细胞动作电位复极3期的主要离子通道,对维持和决定动作电位时程的长短具有重要意义。最近报道在一日本遗传性LQTS家系中发现了hERG基因的一个新的突变位点Q738X,对该基因突变导致hERG钾通道功能的改变迄今国内外尚未见报道。获得性LQTS主要是由于药物引起,药物主要通过两种机制作用于hERG钾通道：①直接抑制hERG钾通道,药物多与hERG钾通道S6区域的两个芳香族氨基酸Y652和F656具有高的亲和力,引起心肌细胞复极3期K+外流减少；②影响hERG蛋白的运输过程,造成细胞膜表面的hERG蛋白数量减少。丙泊酚(propofol)又名异丙酚,是一种快速强效的全身麻醉剂,由丙泊酚引起的心电图QT间期延长,导致发作性晕厥、心脏猝死的心律失常事件时有发生。关于丙泊酚对QT间期影响的报道结果不一,一些研究认为丙泊酚能延长QT间期,相反,有些报道认为丙泊酚不影响甚至缩短QT间期。然而,在这些报道中丙泊酚常和其它药物同时使用,很难判定是丙泊酚单独作用的结果。我们假设丙泊酚对QT间期的影响及其机制可能与hERG钾通道的抑制有关。因此,本实验目的：①突变体Q738X-hERG的功能鉴定；②探讨丙泊酚对野生型(wide type, WT) hERG钾通道和共转染型WT/Q738X-hERG钾通道的影响；③丙泊酚对hERG钾通道作用机制的探讨,为临床合理化和个体化用药提供理论依据。方法1.PCR定点突变Q738X、Y652A和F656C-hERG:采用PCR定点突变技术进行Q738X,Y652A和F656C突变,经DNA测序验证突变成功。2.细胞培养和瞬时转染：人胚肾上皮细胞(human embryonic kidney cell line,HEK-293细胞)在含10%胎牛血清的高糖DMEM培养基中培养。采用磷酸钙沉淀或脂质体介导的方法进行瞬时转染。根据实验要求,转染质粒包括：①野生型WT-hERG;②突变型Q738X、Y652A和F656C-hERG;③共转染型：WT/Q738X-hERG;④对照组WT-hERG和空白质粒PBI。3.全细胞膜片钳记录hERG钾通道电流：寻找表达有绿色荧光的单个HEK-293细胞,采用全细胞膜片钳技术,选用不同的实验方案,分别记录野生型WT、突变型Q738X、共转染型WT/Q738X和对照组的hERG钾通道电流和其动力学特征变化,以及丙泊酚对野生型WT、突变型Y652A和F656C和共转染型WT/Q738X-hERG钾通道电流和其动力学特征的变化。4.Western blot检测hERG蛋白的表达：酶解收集蛋白,BCA法测蛋白含量,RIPA裂解蛋白,10%的SDS-PAGE凝胶电泳,PVDF转膜,5%脱脂牛奶室温封闭,抗hERG抗体(1：200),4℃孵育过夜,HRP标记的二抗(1：10000),室温孵育1 h,ECL显色。通过Quantity one软件分析每条带的灰密度值,与自身内参β-actin条带的灰密度相比,对每条带进行半定量分析。5.双重细胞免疫荧光：分别观察转染WT、Q738X和WT/Q738X-hERG的HEK-293细胞上hERG蛋白的定位情况以及丙泊酚对WT-hERG蛋白定位的影响,转染36～48 h后4%多聚甲醛固定细胞,0.1%Triton X-100透化,2%牛血清白蛋白封闭,随后加入兔源抗hERG蛋白抗体和鸡源抗肌钙网蛋白抗体,4℃孵育过夜,次日加入FITC标记的山羊抗兔IgG和Alexa fluor 633标记的山羊抗鸡IgG的二抗,封片剂封片。6.激光共聚焦技术将细胞免疫荧光染色的HEK-293细胞进行激光共聚焦定位,FITC(绿色)和Alexa fluor(红色)分别用波长488 nm的氩离子激光和633 nm的氦氖离子激光激发。7.统计学处理所有数值用mean±S.E.M表示,使用pulse 8.67软件记录和分析通道电流,采用SPSS13.0和Origin6.0软件来拟合曲线和制图,加药前后采用配对t检验,组间比较采用单因素方差分析。P<0.05为差异有显著性。1.突变体Q738X-hERG的功能鉴定1.1突变体Q738X-hERG电流的记录将转染有野生型WT-hERG的HEK-293细胞进行电生理记录,显示出典型的hERG钾电流,由时间依赖性电流和尾电流两部分组成。而在单独表达有突变型Q738X-hERG的HEK-293细胞中记录不到电流。将WT-hERG和Q738X-hERG(各2μg)共转染到HEK-293细胞中时,虽可记录到hERG钾电流,其电流图形也与WT-hERG电流相似,但其电流幅度明显低于WT-hERG (4μg)而与WT-hERG (2μg)电流大小相似。WT-hERG (4μg)、WT-hERG (2μg)和WT/Q738X(各2μg)的尾电流幅度依次为59.94±3.0、32.2±1.7(P<0.05vs 4μg WT-hERG)和26.2±3.5 pA/pF(P<0.05 vs 4μg WT-hERG);通过对WT/Q738X的动力学观察,发现Q738X突变不改变hERG钾通道的动力学特征(包括激活、失活和去活过程)。1.2突变体Q738X的运输障碍Western blot结果显示转染野生型WT-hERG的HEK-293细胞中可以检测到二条蛋白质条带(135和155 KDa),而突变体Q738X-hERG只出现135 KDa一个条带,共转染型WT/Q738X-hERG虽出现135和155 KDa二条蛋白质条带,但其155KDa的条带较弱。通过Quantity one软件分析每条带的灰密度值,对每条带进行半定量分析。结果显示,共转染WT/Q738X-hERG虽有成熟的hERG蛋白(155 KDa)的形成,但是表达量少于WT-hERG(P<0.01)。说明Q738X的突变影响hERG蛋白的运输,使其到达细胞膜表面有功能的hERG蛋白数量减少。双重细胞免疫荧光染色后经激光共聚焦定位显示：转染有WT-hERG的HEK-293细胞,其绿色荧光主要分布在细胞膜上,与ER marker(红光)共定位后,虽有部分重叠,但膜上的绿色荧光仍然很清楚；而转染有突变体Q738X-hERG的HEK-293细胞,其绿色荧光主要分布在胞浆中,与ER marker共定位后,完全重合,说明突变体Q738X-hERG蛋白分布在内质网中；当将WT和Q738X共转染到HEK-293细胞时,虽在胞浆处有部分重叠,但膜上仍有绿色荧光。2.丙泊酚对WT-hERG钾通道的影响2.1丙泊酚对WT-hERG钾通道的直接影响丙泊酚浓度依赖性的抑制WT-hERG钾通道,当浓度为0.01、0.1、1、3、10、30、100、300、1000和3000μM时,丙泊酚对WT-hERG钾通道的抑制率依次为3.8±2.4%、13.9±7.9%、17.0±5.5%、23.2+5.0%、29.8±4.9%、43.7±5.4%、58.2±4.8、66.4±7.5%、71.2±4.3%和84.3+4.1%,丙泊酚抑制WT-hERG钾通道的IC50值为60.9±6.4μM；使用“尾电流包裹”的实验方法观察10μM丙泊酚对WT-hERG钾通道的时间依赖性,结果显示,随着刺激时程的延长,丙泊酚对WT-hERG钾通道尾电流的抑制程度增加,但大约在1500 ms之后,抑制率达到43.6±3.8%不再有明显增加,说明丙泊酚对WT-hERG钾通道的抑制具有时间依赖性。观察10μM丙泊酚对WT-hERG钾电流的频率依赖性,在频率为1 Hz时,加药后对WT-hERG钾电流的抑制率为71.0±2.1%；在频率为0.2 Hz时,其抑制率为69.4±2.3%。尽管频率不同,但加药后的电流抑制率经统计学处理,无显著差异(P>0.05),说明丙泊酚对WT-hERG钾电流的抑制无频率依赖性。通过观察10μM丙泊酚对WT-hERG钾通道动力学的影响,表明10μM丙泊酚对其动力学特性(包括激活、失活和去活)均无影响。为了解丙泊酚和hERG基因S6区域的两个位点Y652和F656是否具有高亲和力,本实验观察了丙泊酚对突变体Y652A和F656C的作用,结果显示,300μM的丙泊酚对WT-hERG钾通道的抑制率为66.3±7.5%,而对Y652A-hERG钾通道的抑制率仅为21.3±4.1%(P<0.05 vs WT-hERG),丙泊酚抑制Y652A-hERG钾通道电流的IC50值为2871.8±351.9μM；当丙泊酚的浓度为100、300、1000和3000μM时,其对WT-hERG钾通道电流分别抑制了51.0±5.2%、62.4±5.9%、88.4±3.3%和94.7±2.1%；对F656C-hERG钾通道电流分别抑制了8.3±4.5%、21.2±6.3%、39.5±6.7%和45.0±8.3%(P<0.05 vs WT-hERG),可见Y652A和F656C的突变均削弱了丙泊酚抑制WT-hERG钾通道的能力。2.2丙泊酚对WT-hERG蛋白运输的影响经Western blot分析和激光共聚焦结果显示丙泊酚对WT-hERG蛋白的表达和定位无影响。3.丙泊酚对WT/Q738X-hERG钾通道的影响丙泊酚浓度依赖性的抑制WT/Q738X-hERG钾通道,当浓度为O.1、1、3、10、30、100和1000μM时,丙泊酚依次对WT/Q738X-hERG钾通道的抑制率为2.9±4.9%、11.6±3.8%、33.7+3.2%、48.9±2.5%、55.8±3.8%、80.1±2.9%和87.5±2.9%,丙泊酚抑制WT/Q738X-hERG钾通道的IC50值为14.2±2.8μM,但丙泊酚不改变WT/Q738X-hERG钾通道的激活和失活特性。1.突变体Q738X可导致hERG蛋白功能的丧失,对野生型WT-hERG钾通道无负显性抑制,属于运输障碍；2.丙泊酚可直接抑制WT-hERG电流,具有浓度依赖性和时间依赖性,而无频率依赖性,对其动力学特征(激活、失活和去活)无明显影响；丙泊酚与位点Y652和F656有高亲和力；丙泊酚对WT-hERG蛋白的运输无影响；3.Q738X突变可增强丙泊酚对hERG蛋白的抑制作用。更多还原

【Abstract】 There are two types of long QT syndrome (LQTS), inherited and acquired LQTS. Inherited LQTS is caused by mutation of genes encoding cardiac ionic channels or associated partners, which leads to dysfunction of corresponding ionic channel proteins. So far, molecular genetic studies have discovered at least ten forms of inherited LQTS numbered in order of discovery as LQT1-LQT10. Mutation in the hERG gene accounting for LQT2 is one of the principal causes of inherited LQTS. The human ether-a-go-go-related gene (hERG) encodes the a-subunit of the rapidly activating delayed rectifier potassium ion channel underlying IKr-and this, current is essential to the repolarization phase of cardiac action potential in the mammalian heart. More recently, Yasuda et al. reported a small family of hereditary LQT2 caused by a novel hERG mutation, showing a wide variety of ECG phenotypes among family members under the common single nucleotide mutation (c.C2212T and p.Q738X). However, the mechanism for hERG channel dysfunction in the Q738X mutation has not been studied.Acquired LQTS is mainly induced by drugs. The drug-induced LQTS can result from:1. direct block of channel conduction. The drug has high affinity with the particular aromatic amino acid (position Y652 and F656) located on the S6 transmembrane domain of the hERG channel. When it binds with the hERG channel, it leads to K+ efflux decrease in phase 3 repolarization of cardiac cells, and therefore prolongs the length of QT interval.2. Indirect inhibition by disrupting channel protein trafficking. The drug disrupts normal hERG channel protein processing and maturation to reduce its surface membrane expression.Propofol is a short-acting intravenous anesthetic agent widely used for the induction and maintenance of general anesthesia and for sedation in intensive care units. Despite its commendable record, there have been scattered reports of an association of propofol use with sudden death. Several recent studies investigated the effect of poprofol on QT interval, however these results were conflicting. Some studies reported that propofol prolonged the QT interval. Conversely, other studies found that propofol had no effect or shortened the QT interval. In these studies propofol was administered simultaneously with other agents within a short period of time; therefore, it is difficult to determine its selective effect on the QT interval. To our knowledge, the effect and mechanism of poprofol on the QT interval are not entirely clear but could involve actions on the hERG K+ channel.So, the objectives of this study are as follows:1. To examine the electrophysiological consequences of the Q738X mutation; 2. To evaluate the effect of propofol on reconstituted wild type (WT) and its underlying mechanism; 3. To evaluate the effect of propofol on mutant hERG channels using the heterologous expression system in HEK-293 cells.Mutant Q738X, Y652A and F656C-hERG were constructed by overlap extension PCR and were verified by DNA sequencing, respectively.HEK-293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in a humidified 5% CO2 incubator at 37℃.For transfection of hERG constructs in different amounts, cells were plated in a culture dish and transfected transiently 24～36 h later with the calcium phosphate precipitation method or lipofectamine method. Transfected cells were transferred to a bath mounted on the stage of an inverted microscope. First, we studied the current, current-voltage relation, actvation, deactivation and inactivation of WT-hERG and WT/Q738X-hERG channel. Secondly, we observed the effect of propofol on WT-hERG, Y652A, F656C, and WT/Q738X-hERG channel, respectively.The cells were scrapped in ice-cold phosphate-buffered saline and lysed in RIPA buffer. Protein concentrations were determined by the bicinchoninic acid method. Protein per sample was electrophoresed on 10% Tris-acetate gels and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk before incubation with rabbit anti-hERG antibody (1:200) overnight. The membranes were then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10000) in TBST for 1 h at room temperature. After washing with TBST, the membranes were developed using ECL. Blots were analyzed and quantified by Quantity one software.At 36～48 h after transfection, HEK293 cells were fixed with 4% paraformaldehyde, treated with 0.1% Triton X-100, blocked with 2% bovine serum albumin (BSA) at room temperature. Cells were then labeled with rabbit polyclonal anti-hERG and chicken polyclonal anti-calreticulin at 4℃overnight followed by incubation with FITC-conjugated goat antirabbit IgG secondary antibody and Alexa fluor-conjugated goat antichicken IgG secondary antibody at 37℃for 2 h.Immunofluorescence staining was viewed with a confocal laser scanning microscope (excitation and emission wavelength for FITC were 488 and 520nm, respectively; excitation and emission wavelength for Alexa fluor were 633 and 647 nm, respectively).Data are presented as mean±S.E.M. The data were acquired with use of Pulse 8.67 software and analyzed by SPSS 13.0 and Origin 6.0 software. Statistical comparisons were evaluated by t tests and one-way ANOVA. P values of less than 0.05 were taken as significant. There were classic currents from HEK-293 cells transfected with WT-hERG, but no currents from cells transfected with Q738X-hERG channel. These cells cotransfected with WT/Q738X-hERG showed currents with similar waveforms but reduced current amplitudes compared to those cells expressing WT-hERG channels alone. The peak tail current of WT-hERG (4μg), WT-hERG (2μg) and WT/Q738X (2μg each) was 59.9±3.2 pA/pF,32.2±1.7 pA/pF and 26.2±3.5 pA/pF (P<0.05 vs 4μg WT-hERG), respectively. Therefore, we conclude the Q738X mutation had no a potent dominant-negative effect on WT-hERG channel properties.Mutant Q738X had not altered the kinetic features of WT-hERG channel (activation, deactivation and inactivation).The results of Western blot showed two bands (135 and 155 KDa) in WT-hERG group, only one 135 KDa band in Q738X-hERG group, and both bands, but a weaker 155 KDa band in WT/Q738X-hERG group.The results of confocal imaging showed WT-hERG mainly expressed in membrane, Q738X-hERG expressed in cytoplasm and WT/Q738X-hERG expressed in both membrane and cytoplasm.Propofol inhibited WT-hERG channel current in a concentration-dependent manner. When the concentrations of propofol were 0.01,0.1,1,3,10,30,100,300,1000, and 3000μM, the inhibition rates of WT-hERG channel current were 3.8±2.4%, 13.9±7.9%,17.0±5.5%,23.2±5.0%,29.8±4.9%,43.7±5.4%,58.2±4.8%,66.4±7.5%, 71.2±4.3%, and 84.3±4.1%, respectively. The half maximal inhibitory concentration (IC50) of WT-hERG was 60.9±6.4μM.An "envelope of tails" protocol was used to investigate the time-dependence of propofol block. A significant block was achieved with a 50 ms depolarizing step after application of propofol. The extent of block was further increased with increasing pulse duration and maximal block was achieved at 1500 ms, demonstrating the inhibitory effect of propofol on WT-hERG current was time-dependent.The frequency dependence of WT-hERG current block was investigated by applying 30 repetitive pulses at 0.2 and 1 Hz. For control conditions, the HERG current amplitude during the pulse train decreased only slightly. Following exposure to 10μM propofol, application of the pulse train at either 0.2 or 1.0 Hz decreased current amplitude by 69.4±2.3% at 0.2 Hz and by 71.0±2.1% at 1 Hz (P>0.05). Thus, the effect of propofol on WT-hERG had no frequence-dependence.The block of WT-hERG by propofol had not altered the kinetic features of WT-hERG channel (activation, deactivation and inactivation).At 300μM concentration, propofol inhibited the WT-hERG channel by 66.3±7.5% while inhibited Y652A-hERG channel by 21.3±4.1%(P<0.05 vs WT-hERG). The IC50 value of Y652A-hERG was 2871.8±351.9μM. When the concentrations of propofol were 100,300,1000 and 3000μM, the inhibition rates of WT-hERG channel were 51.0±5.2%,62.4±5.9%,88.4±3.3%, and 94.7±2.1%, respectively, and the inhibition rates of F656C-hERG channel were 8.3±4.5%,21.2±6.3%,39.5±6.7%, and 45.0±8.3%, respectively (P<0.05 vs WT-hERG). Mutations in drug-binding sites (Y652A or F656C) of the hERG channel significantly attenuated the hERG current blockade by propofol.The results of Western blot and confocal imaging showed that propofol can not affect the protein trafficking of WT-hERG channel.Propofol blocked WT/Q738X-hERG channel in a concentration-dependent manner. When the concentrations of propofol were 0.1,1,3,10,30,100 and 1000μM, the inhibition of WT/Q738X-hERG were 2.9±4.9%,11.6±3.8%,33.7±3.2%,48.9±2.5%, 55.8±3.8%,80.1±2.9%, and 87.5±2.9%, respectively. The IC50 value of WT/Q738X-hERG was 14.2±2.8μM.The block of WT/Q738X-hERG by propofol had not altered the kinetic features of WT-hERG channel (activation, inactivation and deactivation).1. The Q738X mutation of hERG is nonfunctional and has no a dominant-negative effect on WT-hERG current function;2. Propofol inhibits WT-hERG channel in a concentration-and time-dependent manner, but not frequence-dependent; Propofol have not altered the kinetic features of WT-hERG channel (activation, deactivation, and inactivation); Mutations in drug-binding sites (Y652A or F656C) of the hERG channel attenuate the hERG current blockade by propofol; Propofol does not influence the protein trafficking of WT-hERG channel;3. Mutant Q738X can increase the inhibition of propofol on WT-hERG.更多还原