The Electrophysiological Mechanisms Of Atrial Tachycardia Induced By Potassium Channel Gene KCNQ1 Mutation

Background:Atrial tachycardia(AT)is a rapid arrhythmia with atrial rhythm≥100bpm that occurs in the atria.It can be classified into autonomic AT,reentry AT and trigger active AT according to the different pathogenesis and Electrocardiogram manifestations,and AT with different electrophysiological mechanisms presents different characteristics.Although the genetic basis of AT is not yet clear throughout domestic and abroad studies,AT is often preceded or concomitant atrial fibrillation,and a large number of studies have found that atrial fibrillation is closely related to gene mutation encoding the sodium,potassium and calcium plasma channel.In which potassium channel genes were the most common including KCNQ1,KCNE1,KCNE2 and etc.In cardiomyocytes,the potassium channel alpha subunit encoded by the KCNQ1 gene combine with the Beta subunit encoded by the KCNE1 gene to form a channel complex,which together constitute delayed rectified potassium current(IKs),which play a key role in maintaining cardiac repolarization reserve.Previous studies have found that KCNQ1 gene mutations are closely related to a variety of inherited arrhythmias(such as long QT syndrome,short QT syndrome,atrial fibrillation,etc.).The expression and regulation abnormality of KV7.1 caused by mutations is an important molecular biological basis for arrhythmia.Our team’s previous study screened the potassium channel KCNQ1-V162 M mutation in a patient with atrial tachycardia,but its role in atrial tachycardia and specific pathogenic mechanism are unclear.Objective:The purpose of this study was to investigate the effect of KCNQ1 gene mutation(V162M)on the electrophysiological function of potassium channels.Methods:(1)Plasmid construction: the wild-type(WT)and the mutant(V162M)plasmid of KCNQ1 and KCNE1 plasmid were constructed respectively.The KCNQ1-V162 M mutant plasmid was constructed by gene site-directed mutation induction technique using the KCNQ1-WT plasmid as the template.Meanwhile,primers were designed according to the gene mutation site,and plasmid construction was verified by DNA sequencing.(2)Cell transfection: using liposomes as the medium,KCNQ1-WT and KCNQ1-V162 M mutant plasmids were respectively co-transfected with KCNE1 plasmid to human embryonic kidney 293 cells(HEK293)and African green monkey kidney cells(COS7),and the transfected successfully result was obtained by fluorescence microscopy.(3)Cellular Electrophysiology: after the transfection of wild-type and mutant plasmids with COS7 cells for 48 hours,whole-cell patch clamp technique was used to detect the potassium ion channel function and analyze the electrophysiological characteristics of the two groups of cells.(4)Real-time PCR and Western Blot analysis: After HEK293 cells were transfected with wild-type and mutant plasmids for 48 hours,total RNA,total proteins,membrane proteins and cytoplasmic proteins of the two groups of cells were extracted,and Rt-PCR and Western blotting were applied to analyze the KCNQ1 m RNA and channel proteins expression level of the two groups.Results:(1)Cell electrophysiological studies showed that compared with WT group,the IKs peak current density and the tail current density of V162 M mutant group were respectively decreased significantly when the voltage was 20～140m V and40～120m V.(2)The results of Rt-PCR and western blotting showed that KCNQ1 gene m RNA,total protein and membrane protein expression in V162 M mutant group were all decreased compared with WT group.Conclusion:(1)KCNQ1-V162 M mutation decreased potassium channel function,which may be the electrophysiological mechanism of the occurrence of atrial tachycardia caused by this mutation.(2)Decreased expression of KCNQ1 channel membrane protein may be one of the pathological mechanisms.