钠离子通道功能改变在遗传性长QT综合征发病机制中的研究
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摘要
背景
遗传性长QT综合征(long QTsyndrome, LQTS)是一类遗传性的心律失常疾病,临床表现以反复发作的晕厥、抽搐甚至猝死为特征。体表心电图上表现为QT间期延长、T波异常(T波低平或双相),易发生室性快速心律失常(ventricular tachyarrhythmia, VT)或尖端扭转型室性心动过速(Torsade de Pointes, TdP),可能转化为心室颤动(ventricular fibrillation,VF)等恶性心律失常。长QT综合征是心脏结构正常、有不明原因晕厥的年轻人发生猝死的常见原因之一。
流行病学研究显示,大多数遗传性长QT综合征具有家族聚集性,只有极少数是由于散在的新发突变引起。迄今为止共发现了13个基因的数千种基因变异导致的13种不同类型的长QT综合征,其中6种基因(KCNQ1、KCNH2、SCN5A、KCNJ2、 CACNA1C、KCNJ5)编码离子通道的结构蛋白,另外7种基因(ANKB、KCNE1、KCNE2、 CAV3、SCN4B、AKAP9、SNTA1)编码离子通道的调节蛋白。
SCN5A基因编码人类心肌细胞上的电压门控性钠离子通道(hNav1.5)的α亚单位,α亚单位是完成钠离子通道功能的主要结构,可产生较大的内向钠离子电流,引起心肌细胞动作电位快速上升,同时使冲动在心肌组织间快速传导。该通道蛋白由位于胞内的N端、位于胞内的C端和4个同源结构域(DI-DIV)组成,每个同源结构域包含6个跨膜节段(S1-S6)。SCN5A基因发生突变可引起钠离子通道“功能增强”,延长心肌细胞动作电位时程(action potential duration, APD),延长QT间期,从而导致长QT综合征3型(LQT3).
α1-互生蛋白(a1-syntrophin, SNTA1)属于抗肌萎缩蛋白(dystrophin)相关的蛋白家族,是互生蛋白(syntrophin)在骨骼肌和心肌细胞中的主要亚型。作为适配体,SNTA1蛋白可与神经性一氧化氮合酶(neuronal nitric oxide synthase, nNOS)、细胞膜钙ATP酶(plasma membrane Ca2+/calmodulin-dependent ATPase, PMCA4b)相互结合,构成复合体。在此复合体中,PMCA4b可抑制nNOS的功能,减少一氧化氮(NO)的合成。SNTA1还可直接通过PDZ结构域的结合序列直接与hNav1.5C端的最后三个氨基酸残基——丝氨酸-异亮氨酸-缬氨酸(serine-isoleucine-valine)相互作用。既往研究在长QT综合征患者中发现SNTA1基因的一个突变A390V可以破坏SNTA1与PMCA4b的结合作用,拮抗PMCA4b对nNOS的抑制作用,显著增加INa的峰电流和晚钠电流大小,标志着SNTA1成为长QT综合征12型(LQT12)的致病基因。
目的
定位一个长QT综合征家系的致病基因；识别基因突变位点；在细胞水平上研究该突变对其编码蛋白质功能的影响,探讨基因突变导致长QT综合征的发病机制。
方法
收集长QT综合征家系家族成员的外周静脉血,采用候选基因策略对目前已发现的13个长QT综合征的致病基因的全部外显子和外显子-内含子结合部位进行筛查,以期发现致病基因。在野生型质粒的基础上,采用定点突变技术构建突变体质粒,并利用瞬时转染的方法在异源真核生物表达系统——人胚肾293细胞(HEK293)中表达。采用全细胞膜片钳技术在转染了野生型或突变型质粒的HEK293细胞上记录通道电流,研究相应通道的电生理特性。
结果
先证者,女性,37岁,反复发作性晕厥30年,12导联体表心电图示校正的QT间期(QTc)延长(大于480ms)。先证者10岁的儿子因多次发生阵发性室上速接受过射频消融术治疗,体表心电图示QTc=500ms。其他家庭成员无明显症状。基因型分析发现,先证者的SCN5A基因和SNTA1基因存在两个新的基因变异,均引起该位点编码的氨基酸发生改变,分别为R800L和A261V。利用全细胞膜片钳技术在表达了野生型和突变体钠离子通道的HEK293细胞上记录钠电流(sodium current,IN。),发现与野生型通道(WT-SCN5A+WT-SNTA1)相比,双突变体通道(R800L-SCN5A+A261V-SNTA1)和单突变体通道(R800L-SCN5A+WT-SNTA1, WT-SCN5A+A261V-SNTA1)的晚钠电流(late sodium current,late/Na)增加,电流衰减减慢,以双突变体通道的改变最为显著。这一钠离子通道“功能增强”效应可被nNOS的抑制剂N-单甲基-L-精氨酸(NG-monomethyl-L-arginie, L-NMMA)所阻断。
结论
分别位于SCN5A基因和SNTA1基因的两个新的基因突变R800L与A261V通过影响钠离子通道的亚硝基化作用,共同导致钠离子通道“功能增强”效应,增加晚钠电流,减慢电流衰减,延长了动作电位时程,从而引起长QT综合征。
背景
SCN5A基因编码人类心肌细胞电压门控性钠离子通道(hNav1.5)的α亚单位,α亚单位是完成钠离子通道功能的主要结构,可产生较大的内向钠离子电流(sodium current,/Na),引起心肌细胞动作电位快速上升,同时使冲动在心肌组织间快速传导。α亚单位还与晚钠电流(late sodium current, late/Na)的产生有关,后者可影响心肌细胞的复极过程。SCN5A基因发生突变可引起一系列遗传性心律失常疾病,如长QT综合征3型、Brugada综合征、心脏传导系统疾病、病态窦房结综合征等。钠离子通道的功能异常往往与SCN5A基因的选择性剪接变异相关,并受到环境物理化学因素如酸中毒的影响。既往报道SCN5A基因的一个错义突变S1787N可引起长QT综合征3型,这一突变也在295名健康白人对照里发现了一例。
目的
本研究拟在SCN5A选择性剪接变异和不同pH条件的背景下,研究S1787N-SCN5A突变对钠离子通道电生理功能的影响,揭示该突变的致病机制。
方法
SCN5A基因有两种常见的剪接异构体,一种在第1077位氨基酸上缺失一个谷氨酸,记做Q1077del,是存在最多的剪接异构体；另一种则在第1077位氨基酸上未发生谷氨酸的缺失,记做Q1077。我们将S1787N突变构建于SCN5A基因这两种剪接异构体中,利用瞬时转染的方法在异源真核生物表达系统——人胚肾293细胞(HEK293)中表达。转染后24小时,应用全细胞膜片钳技术在转染了野生型或突变型钠离子通道的HEK293细胞上记录钠离子电流,研究钠离子通道的电生理特性。本实验中,我们采用了不同pH值的细胞内液。
结果
Q1077剪接变异体背景下的S1787N突变,钠离子通道的峰值电流密度(peak/Na density)、稳态激活过程和稳态失活过程的动力学参数以及晚钠电流的相对值(percentage of/Na late/peak)与野生型钠离子通道相比,无明显差异,改变细胞内液的pH值并不影响通道的电生理特性。而Q1077del剪接变异体背景下的S1787N突变,当细胞内液pH值正常即pH=7.4时,晚钠电流的相对值是野生型钠离子通道的2.1倍,统计学上有显著性差异(P<0.05)；当细胞内液pH值下降即pH=6.7时,晚钠电流的相对值是野生型钠离子通道的2.9倍,统计学上有显著性差异(P<0.03)。
结论
致长QJ综合征3型的突变S1787N-SCN5A在不同SCN5A剪接变异体和不同细胞内pH值的环境下,呈现出不同的电生理特性。这一发现表明SCN5A基因的选择性剪接方式和物理化学环境因素均可对钠离子通道的功能产生影响,从而引起不同的分子表型和临床表型。
Background
The inherited long QT syndrome (LQTS) is a hereditary cardiac disorder characterized by prolonged QT interval on the surface electrocardiogram (ECG) and increased risk for sudden death due to ventricular tachyarrhythmia. It is one of the common causes of unexplained syncope especially in young, seemingly healthy individuals. To date more than1000mutations in13genes have been identified to associate with LQTS, in which6genes encode cardiac ion channels (KCNQ1, KCNH2, SCN5A, KCNJ2, CACNA1C and KCNJ5), and7encode ion channel subunits or channel-interacting proteins (ChIPs)(ANKB, KCNE1, KCNE2, CAV3, SCN4B, AKAP9and SNTA1).
SCN5A encodes the α subunit of voltage-gated cardiac sodium channel hNav1.5also denoted SCN5A that is responsible for large peak inward sodium current (/Na) in the heart. The a subunit has four homologous domains (DI-DIV) and each domain contains six transmembrane segments (S1-S6). LQTS-associated mutations in SCN5A cause LQT3, a "gain-of-function" leading to prolonged cardiac action potential duration, lengthened QT interval, and increased risk of arrhythmia.
a1-syntrophin (SNTA1), a dystrophin-associated protein, is the dominant syntrophin isoform in skeletal and cardiac muscle. As a scaffolding adapter, SNTA1binds to neuronal nitric oxide synthase (nNOS), which is constitutively expressed in the heart, and the cardiac isoform of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA4b) to form a complex in which PMCA4b acts as a potent inhibitor of NO synthesis. SNTA1also interacts directly with the PDZ domain-binding motif formed by the last three residues (serine-isoleucine-valine) of Navl.5C terminus. Previous study revealed a novel mutation A390V-SNTA1in a LQTS patient disrupted the association with PMCA4b and antagonized the inhibition of nNOS, resulting in augmentation of both peak and late/Na, suggesting SNTA1as a novel LQTS-susceptibility gene (LQT12).
Objectives
The present study was designed to identify potentially novel mutations which would be responsible for LQTS in a Caucasian pedigree, to further characterize electrophysiological features and to elucidate a plausible pathogenic arrhythmia mechanism for LQTS.
Methods
A Caucasian family with syncope and prolonged QT interval was identified. Genomic DNA was extracted from peripheral blood lymphocytes and was screened for the entire open-reading frames of13LQTS-susceptibility genes by polymerase chain reaction (PCR), denaturing high-performance liquid chromatography (HPLC) and direct DNA sequencing. Identified mutations were created with the aid of site-directed mutagenesis. Wild-type or mutant channels were heterologously expressed in HEK293cells by transient co-transfection. Macroscopic voltage-gated/Na was measured24hours after transfection with the standard whole-cell patch clamp technique in HEK293cells.
Results
The proband was a37-year-old woman who had experienced recurrent bouts of syncope for30years. Her resting12-lead ECG showed a marginal prolongation of corrected QT interval (QTc>480ms). Her10-year-old son was suffered from supraventricular tachycardia and the ECG showed moderately prolonged QTc (500ms). Genetic analysis revealed they both harbored the R800L mutation in SCN5A and A261V mutation in SNTA1. Other family members harboring either mutation had weaker clinical phenotype. Functional assays showed Peak/Na densities were unchanged for WT and for mutant channels containing R800L-SCN5A, A261V-SNTA1or R800L-SCN5A plus A261V-SNTA1. However, late/Na for either single mutant was moderately increased2-3fold compared to WT. The combined mutations of R800L-SCN5A plus A261V-SNTA1significantly enhanced the/Na late/peak ratio by5.6-fold compared with WT. The time constants of current decay of combined mutant channel were markedly increased. The "gain-of-function" effect could be blocked by the NG-monomethyl-L-arginine (L-NMMA), a nNOS inhibitor.
Conclusions
We conclude that novel mutations in SCN5A and SNTA1jointly exert a nNOS dependent "gain-of-function" on SCN5A channels (i.e., increased late/Na as well as slowed current decay), which may consequently prolong the action potential duration and lead to LQTS phenotype.
Background
SCN5A encodes the voltage-dependent sodium channel α-subunit protein SCN5A, also called hNav1.5, found predominantly in human heart muscle. This channel is responsible for large peak inward sodium current (/Na) that underlies excitability and conduction in working myocardium (atrial and ventricular cells) and special conduction tissue (Purkinje cells and others), and also for late/Na that influences repolarization and refractoriness. SCN5A in humans has two splice variants, one lacking a glutamine at position1077(Q1077del) and one containing Q1077. Mutations in SCN5A can cause a broad variety of pathophysiological phenotypes, such as long QT syndrome type3(LQJ3), Brugada syndrome (BrS), cardiac conduction disease (CCD), or sick sinus syndrome (SSS)./Na dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed, and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in1of295healthy white controls.
Objectives
Here, we determined the in vitro biophysical phenotype of S1787N-SCN5A in an effort to further assess its possible pathogenicity.
Methods
We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position1077(Q1077del) and the minor isoform which contains Q1077in the pcDNA3.1vector, and expressed them in HEK293cells for eletrophysiological study. Macroscopic voltage-gated/Na was measured24hours after transfection with the standard whole-cell patch clamp technique. We applied two kinds of intracellular solution varied in pH.
Results
After24h transfection, S1787N in Q1077background had WT-like/Na including peak/Na density, activation and inactivation parameters as well as late/Na in both pHi7.4and pHi6.7. However, with S1787N in the Q1077del background, the percentage of/Na late/peak was increased2.1fold compared to WT in pHi7.4(n=7-9, p<0.05) and was increased2.9fold compared to WT in pHi6.7(n=6-8, p<0.03).
Conclusions
A LQT3-like biophysical phenotype for S1787N is both SCN5A isoform and intracellular pH dependent. These findings provide further evidence that the splice variant and environmental factors could affect the molecular phenotype with implications for the clinical phenotype and may provide insight into acidosis-induced arrhythmia mechanisms.
遗传性长QT综合征(long QTsyndrome, LQTS)是一类遗传性的心律失常疾病,临床表现以反复发作的晕厥、抽搐甚至猝死为特征。体表心电图上表现为QT间期延长、T波异常(T波低平或双相),易发生室性快速心律失常(ventricular tachyarrhythmia, VT)或尖端扭转型室性心动过速(Torsade de Pointes, TdP),可能转化为心室颤动(ventricular fibrillation,VF)等恶性心律失常。长QT综合征是心脏结构正常、有不明原因晕厥的年轻人发生猝死的常见原因之一。
流行病学研究显示,大多数遗传性长QT综合征具有家族聚集性,只有极少数是由于散在的新发突变引起。迄今为止共发现了13个基因的数千种基因变异导致的13种不同类型的长QT综合征,其中6种基因(KCNQ1、KCNH2、SCN5A、KCNJ2、 CACNA1C、KCNJ5)编码离子通道的结构蛋白,另外7种基因(ANKB、KCNE1、KCNE2、 CAV3、SCN4B、AKAP9、SNTA1)编码离子通道的调节蛋白。
SCN5A基因编码人类心肌细胞上的电压门控性钠离子通道(hNav1.5)的α亚单位,α亚单位是完成钠离子通道功能的主要结构,可产生较大的内向钠离子电流,引起心肌细胞动作电位快速上升,同时使冲动在心肌组织间快速传导。该通道蛋白由位于胞内的N端、位于胞内的C端和4个同源结构域(DI-DIV)组成,每个同源结构域包含6个跨膜节段(S1-S6)。SCN5A基因发生突变可引起钠离子通道“功能增强”,延长心肌细胞动作电位时程(action potential duration, APD),延长QT间期,从而导致长QT综合征3型(LQT3).
α1-互生蛋白(a1-syntrophin, SNTA1)属于抗肌萎缩蛋白(dystrophin)相关的蛋白家族,是互生蛋白(syntrophin)在骨骼肌和心肌细胞中的主要亚型。作为适配体,SNTA1蛋白可与神经性一氧化氮合酶(neuronal nitric oxide synthase, nNOS)、细胞膜钙ATP酶(plasma membrane Ca2+/calmodulin-dependent ATPase, PMCA4b)相互结合,构成复合体。在此复合体中,PMCA4b可抑制nNOS的功能,减少一氧化氮(NO)的合成。SNTA1还可直接通过PDZ结构域的结合序列直接与hNav1.5C端的最后三个氨基酸残基——丝氨酸-异亮氨酸-缬氨酸(serine-isoleucine-valine)相互作用。既往研究在长QT综合征患者中发现SNTA1基因的一个突变A390V可以破坏SNTA1与PMCA4b的结合作用,拮抗PMCA4b对nNOS的抑制作用,显著增加INa的峰电流和晚钠电流大小,标志着SNTA1成为长QT综合征12型(LQT12)的致病基因。
目的
定位一个长QT综合征家系的致病基因；识别基因突变位点；在细胞水平上研究该突变对其编码蛋白质功能的影响,探讨基因突变导致长QT综合征的发病机制。
方法
收集长QT综合征家系家族成员的外周静脉血,采用候选基因策略对目前已发现的13个长QT综合征的致病基因的全部外显子和外显子-内含子结合部位进行筛查,以期发现致病基因。在野生型质粒的基础上,采用定点突变技术构建突变体质粒,并利用瞬时转染的方法在异源真核生物表达系统——人胚肾293细胞(HEK293)中表达。采用全细胞膜片钳技术在转染了野生型或突变型质粒的HEK293细胞上记录通道电流,研究相应通道的电生理特性。
结果
先证者,女性,37岁,反复发作性晕厥30年,12导联体表心电图示校正的QT间期(QTc)延长(大于480ms)。先证者10岁的儿子因多次发生阵发性室上速接受过射频消融术治疗,体表心电图示QTc=500ms。其他家庭成员无明显症状。基因型分析发现,先证者的SCN5A基因和SNTA1基因存在两个新的基因变异,均引起该位点编码的氨基酸发生改变,分别为R800L和A261V。利用全细胞膜片钳技术在表达了野生型和突变体钠离子通道的HEK293细胞上记录钠电流(sodium current,IN。),发现与野生型通道(WT-SCN5A+WT-SNTA1)相比,双突变体通道(R800L-SCN5A+A261V-SNTA1)和单突变体通道(R800L-SCN5A+WT-SNTA1, WT-SCN5A+A261V-SNTA1)的晚钠电流(late sodium current,late/Na)增加,电流衰减减慢,以双突变体通道的改变最为显著。这一钠离子通道“功能增强”效应可被nNOS的抑制剂N-单甲基-L-精氨酸(NG-monomethyl-L-arginie, L-NMMA)所阻断。
结论
分别位于SCN5A基因和SNTA1基因的两个新的基因突变R800L与A261V通过影响钠离子通道的亚硝基化作用,共同导致钠离子通道“功能增强”效应,增加晚钠电流,减慢电流衰减,延长了动作电位时程,从而引起长QT综合征。
背景
SCN5A基因编码人类心肌细胞电压门控性钠离子通道(hNav1.5)的α亚单位,α亚单位是完成钠离子通道功能的主要结构,可产生较大的内向钠离子电流(sodium current,/Na),引起心肌细胞动作电位快速上升,同时使冲动在心肌组织间快速传导。α亚单位还与晚钠电流(late sodium current, late/Na)的产生有关,后者可影响心肌细胞的复极过程。SCN5A基因发生突变可引起一系列遗传性心律失常疾病,如长QT综合征3型、Brugada综合征、心脏传导系统疾病、病态窦房结综合征等。钠离子通道的功能异常往往与SCN5A基因的选择性剪接变异相关,并受到环境物理化学因素如酸中毒的影响。既往报道SCN5A基因的一个错义突变S1787N可引起长QT综合征3型,这一突变也在295名健康白人对照里发现了一例。
目的
本研究拟在SCN5A选择性剪接变异和不同pH条件的背景下,研究S1787N-SCN5A突变对钠离子通道电生理功能的影响,揭示该突变的致病机制。
方法
SCN5A基因有两种常见的剪接异构体,一种在第1077位氨基酸上缺失一个谷氨酸,记做Q1077del,是存在最多的剪接异构体；另一种则在第1077位氨基酸上未发生谷氨酸的缺失,记做Q1077。我们将S1787N突变构建于SCN5A基因这两种剪接异构体中,利用瞬时转染的方法在异源真核生物表达系统——人胚肾293细胞(HEK293)中表达。转染后24小时,应用全细胞膜片钳技术在转染了野生型或突变型钠离子通道的HEK293细胞上记录钠离子电流,研究钠离子通道的电生理特性。本实验中,我们采用了不同pH值的细胞内液。
结果
Q1077剪接变异体背景下的S1787N突变,钠离子通道的峰值电流密度(peak/Na density)、稳态激活过程和稳态失活过程的动力学参数以及晚钠电流的相对值(percentage of/Na late/peak)与野生型钠离子通道相比,无明显差异,改变细胞内液的pH值并不影响通道的电生理特性。而Q1077del剪接变异体背景下的S1787N突变,当细胞内液pH值正常即pH=7.4时,晚钠电流的相对值是野生型钠离子通道的2.1倍,统计学上有显著性差异(P<0.05)；当细胞内液pH值下降即pH=6.7时,晚钠电流的相对值是野生型钠离子通道的2.9倍,统计学上有显著性差异(P<0.03)。
结论
致长QJ综合征3型的突变S1787N-SCN5A在不同SCN5A剪接变异体和不同细胞内pH值的环境下,呈现出不同的电生理特性。这一发现表明SCN5A基因的选择性剪接方式和物理化学环境因素均可对钠离子通道的功能产生影响,从而引起不同的分子表型和临床表型。
Background
The inherited long QT syndrome (LQTS) is a hereditary cardiac disorder characterized by prolonged QT interval on the surface electrocardiogram (ECG) and increased risk for sudden death due to ventricular tachyarrhythmia. It is one of the common causes of unexplained syncope especially in young, seemingly healthy individuals. To date more than1000mutations in13genes have been identified to associate with LQTS, in which6genes encode cardiac ion channels (KCNQ1, KCNH2, SCN5A, KCNJ2, CACNA1C and KCNJ5), and7encode ion channel subunits or channel-interacting proteins (ChIPs)(ANKB, KCNE1, KCNE2, CAV3, SCN4B, AKAP9and SNTA1).
SCN5A encodes the α subunit of voltage-gated cardiac sodium channel hNav1.5also denoted SCN5A that is responsible for large peak inward sodium current (/Na) in the heart. The a subunit has four homologous domains (DI-DIV) and each domain contains six transmembrane segments (S1-S6). LQTS-associated mutations in SCN5A cause LQT3, a "gain-of-function" leading to prolonged cardiac action potential duration, lengthened QT interval, and increased risk of arrhythmia.
a1-syntrophin (SNTA1), a dystrophin-associated protein, is the dominant syntrophin isoform in skeletal and cardiac muscle. As a scaffolding adapter, SNTA1binds to neuronal nitric oxide synthase (nNOS), which is constitutively expressed in the heart, and the cardiac isoform of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA4b) to form a complex in which PMCA4b acts as a potent inhibitor of NO synthesis. SNTA1also interacts directly with the PDZ domain-binding motif formed by the last three residues (serine-isoleucine-valine) of Navl.5C terminus. Previous study revealed a novel mutation A390V-SNTA1in a LQTS patient disrupted the association with PMCA4b and antagonized the inhibition of nNOS, resulting in augmentation of both peak and late/Na, suggesting SNTA1as a novel LQTS-susceptibility gene (LQT12).
Objectives
The present study was designed to identify potentially novel mutations which would be responsible for LQTS in a Caucasian pedigree, to further characterize electrophysiological features and to elucidate a plausible pathogenic arrhythmia mechanism for LQTS.
Methods
A Caucasian family with syncope and prolonged QT interval was identified. Genomic DNA was extracted from peripheral blood lymphocytes and was screened for the entire open-reading frames of13LQTS-susceptibility genes by polymerase chain reaction (PCR), denaturing high-performance liquid chromatography (HPLC) and direct DNA sequencing. Identified mutations were created with the aid of site-directed mutagenesis. Wild-type or mutant channels were heterologously expressed in HEK293cells by transient co-transfection. Macroscopic voltage-gated/Na was measured24hours after transfection with the standard whole-cell patch clamp technique in HEK293cells.
Results
The proband was a37-year-old woman who had experienced recurrent bouts of syncope for30years. Her resting12-lead ECG showed a marginal prolongation of corrected QT interval (QTc>480ms). Her10-year-old son was suffered from supraventricular tachycardia and the ECG showed moderately prolonged QTc (500ms). Genetic analysis revealed they both harbored the R800L mutation in SCN5A and A261V mutation in SNTA1. Other family members harboring either mutation had weaker clinical phenotype. Functional assays showed Peak/Na densities were unchanged for WT and for mutant channels containing R800L-SCN5A, A261V-SNTA1or R800L-SCN5A plus A261V-SNTA1. However, late/Na for either single mutant was moderately increased2-3fold compared to WT. The combined mutations of R800L-SCN5A plus A261V-SNTA1significantly enhanced the/Na late/peak ratio by5.6-fold compared with WT. The time constants of current decay of combined mutant channel were markedly increased. The "gain-of-function" effect could be blocked by the NG-monomethyl-L-arginine (L-NMMA), a nNOS inhibitor.
Conclusions
We conclude that novel mutations in SCN5A and SNTA1jointly exert a nNOS dependent "gain-of-function" on SCN5A channels (i.e., increased late/Na as well as slowed current decay), which may consequently prolong the action potential duration and lead to LQTS phenotype.
Background
SCN5A encodes the voltage-dependent sodium channel α-subunit protein SCN5A, also called hNav1.5, found predominantly in human heart muscle. This channel is responsible for large peak inward sodium current (/Na) that underlies excitability and conduction in working myocardium (atrial and ventricular cells) and special conduction tissue (Purkinje cells and others), and also for late/Na that influences repolarization and refractoriness. SCN5A in humans has two splice variants, one lacking a glutamine at position1077(Q1077del) and one containing Q1077. Mutations in SCN5A can cause a broad variety of pathophysiological phenotypes, such as long QT syndrome type3(LQJ3), Brugada syndrome (BrS), cardiac conduction disease (CCD), or sick sinus syndrome (SSS)./Na dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed, and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in1of295healthy white controls.
Objectives
Here, we determined the in vitro biophysical phenotype of S1787N-SCN5A in an effort to further assess its possible pathogenicity.
Methods
We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position1077(Q1077del) and the minor isoform which contains Q1077in the pcDNA3.1vector, and expressed them in HEK293cells for eletrophysiological study. Macroscopic voltage-gated/Na was measured24hours after transfection with the standard whole-cell patch clamp technique. We applied two kinds of intracellular solution varied in pH.
Results
After24h transfection, S1787N in Q1077background had WT-like/Na including peak/Na density, activation and inactivation parameters as well as late/Na in both pHi7.4and pHi6.7. However, with S1787N in the Q1077del background, the percentage of/Na late/peak was increased2.1fold compared to WT in pHi7.4(n=7-9, p<0.05) and was increased2.9fold compared to WT in pHi6.7(n=6-8, p<0.03).
Conclusions
A LQT3-like biophysical phenotype for S1787N is both SCN5A isoform and intracellular pH dependent. These findings provide further evidence that the splice variant and environmental factors could affect the molecular phenotype with implications for the clinical phenotype and may provide insight into acidosis-induced arrhythmia mechanisms.
引文
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