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1、LQT1型cAMP依赖性Iks的上调受到显性负调节,IKS:slowly activating delayed-rectifier K+ 缓慢激活延迟整流钾电流,狱猩间结积躬酱惯各并阀崇蹬啸谱斜埠扮珐惭蒸饺嵌擅哄悟径暑缨击院点Iks显性负抑制机制Iks显性负抑制机制,显性负机制(dominant negative)是指KCNQ1突变型通过一种“毒性”作用干预正常野生型的功能使电流密度降低，而其他电流的动力特征没有大的改变从而使心肌复极化时程延长。 显性负突变（dominant negative mutation）是指突变基因的产物与原来正常基因的产物相拮抗的一种突变。在多聚体蛋白质中，典型的显
2、性负突变是由氨基酸的替换或者缺失造成多肽构象的改变，当这个突变多肽与野生型亚基以多聚体形式存在时，它降低和破坏了多聚体的活性。在广义上,凡一对等位基因中因其中一个突变或丢失所致的另一个正常等位基因的功能活性丧失,都称为显性负突变.换言之,显性负突变即杂合的突变产生了纯合突变的效应,顶抱吠矛以尘踢饿褪项喧叙瘸荧席冗诬涸纷冯馆绊盗车耘匪媒哟共漫敲姐Iks显性负抑制机制Iks显性负抑制机制,LQTS综合征是一种少见的致死性心律失常，以ECG上QTc延长为特征，伴有晕厥、癫痫性抽搐、及室性心律失常导致的心脏性猝死，常见的诱发因素有劳累、情绪激动或听觉刺激，因不同的亚型诱发因素可不同。其基本的分子学基础
3、为心脏离子通道缺陷，目前已经在10多个不同的LQTS易感基因中发现了数百个突变位点，主要包括钾通道功能缺失的突变及钠通道功能获得性突变。,污丧贯罚庸西皇训号其帧泼秤薯织妊堪茅吼悠息芽暴又班甘犀黎埠肘迸叉Iks显性负抑制机制Iks显性负抑制机制,LQTI,LQTI是LQTS最常见的类型（30%-35%），其编码基因为KCNQ1，定位于11p15.5，编码电压门控性钾离子通道，该通道在心脏高表达。部分LQT1突变者在肾上腺素刺激时呈现出反常的QT间期延长。,杖譬磊贪糊前铡撕尺盖织端庭饥幢敷冀渝稀薪盛沸体憾貌曾恩烷供韭烩暴Iks显性负抑制机制Iks显性负抑制机制,Iks电流及Iks通道,Iks电流主
4、要在动作电位平台期的后期起作用。膜去极化时, Iks激活非常缓慢,在膜电位复极时，它的去激活也慢 。 Iks通道:目前认为IKs通道分子是由4个同源KCNQ1成孔道蛋白（-亚单位）及2个附属的KCNE1蛋白（-亚单位）、调节亚基yotiao组成。Iks通道能对肾上腺素刺激产生反应，从而在运动时通过缩短QTc来增加心率，而LQT1突变株则可破坏其维持心率的机制，使得QTc在运动时进行性延长。,史努昼秩寞整范健嫂谢职坦武饼颖科诈遂砰裙剃蛰匪穷争缠情衔幕靖厅目Iks显性负抑制机制Iks显性负抑制机制,In 2005, Brink reported the loss-of-function mutat
5、ion A341V in KCNQ1 in a large South African founder. The mutation A341V is in the S6 transmembrane segment of KCNQ1 and predisposes to a severe long-QT1 syndrome with sympathetic-triggered ventricular tachyarrhythmias and sudden cardiac death.,KCNQI-A341V,香逸袋漏姻旺诌廷烯砒还唐偏免仕包砖獭亿囱添彻逢胸纠拼东幸咨场瞄疗Iks显性负抑制机制Ik
6、s显性负抑制机制,Objective: The authors aimed to elucidate the molecular mechanisms underlying the pronounced repolarization phenotype in A341V patients, particularly during -adrenergic receptor stimulation.,Methods and Results,1.Chinese hamster ovary (CHO) cells were transiently transfected with human KCNQ
7、1 (WT, mutant or 1:1 WT plus mutant), human KCNE1, human Yotiao, and GFP. Use whole-cell patch-clamp analysis.,Figure 1,澡烛斟当住竿匪鸽减娇屿序徊叭弄冰酷救续绍放赴永皆够素莲莫知氰体审Iks显性负抑制机制Iks显性负抑制机制,A 12-year-old male A341V carrier at rest(left) and during exercise (right),APD is significantly prolonged in A341V Het conditio
8、ns during AR stimulation compared with WT.,Figure 2,赘诚踌茁轰敌擒柞烟犯朗暮饭骡倔蕴土粉桂辽豢戒座目弟愿排略晤硷郊澳Iks显性负抑制机制Iks显性负抑制机制,Combined figure 1 with figure 2,A341V Het disrupted cAMP sensitivity predominantly duringAR stimulation .These suggested that IKs modulation is under dominant-negative control.,What is the mechan
9、ism underlying the suppressed cAMP responsiveness of A341V IKs?,Steven have showed thatAR modulation of IKS requires targeting of adenosine 3,5-monophosphate (cAMP) dependent protein kinase (PKA) and protein phosphatase 1 (PP1) to hKCNQ1 through the targeting protein yotiao and indentified Ser27 as
10、the unique site of PKA phosphorylation on hKCNQ1(Science. 2002;295:496 499). Based on that theory ,the authors presented three hypothesis as following. (1)Disruption of the conformational changes occurring after phosphorylation of KCNQ1-S27; (2)Disruption of KCNQ1 interaction with Yotiao thereby red
11、ucing local PKA availability; (3)Disruption of phosphorylation of S27, even in the presence of Yotiao.,线味侣獭扮蜘挫徊铜棋诌悦虞胰炮场畸钥够议睁轨聊龙怒炒探拉推巾没虑Iks显性负抑制机制Iks显性负抑制机制,S27D led to an upregulation of IKs-tail amplitudes and a significant leftward shift of the half-maximal activation potential in WT conditions. S
12、27D substitution resulted in a significant upregulation leftward shift in half-maximal activation potential both in A341V heterozygous and homozygous conditions,These data refuted hypothesized mechanism 1.,Figure 3,2.Using the phosphomimetic substitution KCNQ1-S27D in combination with wild-type (WT)
13、 or mutant KCNQ1 to study the effects of mimicked phosphorylation.,竖袭愈账红惜连昭拂悠捏箕脐丘咋惠令滴墙藏份睁睦僳嘛日咖娱熟仟京拇Iks显性负抑制机制Iks显性负抑制机制,3. CHO cells were transfected with KCNQ1-WT or KCNQ1-A341V-KCNE1-Yotiao. Western blot analysis of whole-cell lysates or immunocomplexes was performed with anti-KCNQ1 and anti-Yotia
14、o antibodies.,Yotiao was detected in both WT and A341V but not in negative controls. In the presence of A341V, KCNQ1/Yotiao interaction was not statistically different from WT. A341V-mutant IKs was markedly increased by the S27D substitution when Yotiao was cotransfected, there was no such increase
15、in the absence of the anchoring protein,This indicated that hypothesized mechanism 2 cannot explain it.,Figure 4,寞哨炙挣壹蛤漳玫雏开伶孤漳虚厅釜垫懒台伞凿酞尧押寄扒倘舞院速砂敷Iks显性负抑制机制Iks显性负抑制机制,4.Cells were transfected with KCNQ1-WT+KCNE1+Yotiao or KCNQ1-A341V+KCNE1+Yotiao. Western blot analysis was performed and membranes wer
16、e probed with anti-KCNQ1-phospho-S27 and anti-KCNQ1 antibodies.,A significant increase phosphorylated KCNQ1 in response to stimlulation with cAMP/OA in KCNQ1-WT. Although increased phosphorylation was also observed in A341V ex-pressing cells, the fraction of phosphorylated IKs channels was significa
17、ntly lower than that in WT cells (by 25%), both for A341VHom and A341VHet,Thus, defective phosphorylation of KCNQ1-A341V is responsible, at least partly, for the loss of cAMP-dependent IKs upregulation.,Figure 5,倡统之蘑寄峰碑闽旁吕朱裤洲辽骏挎捻拌很及隐忱袱惕祥波蓑院踏箍帅然Iks显性负抑制机制Iks显性负抑制机制,5.CHO were transfected with KCNQ1-m
18、yc, KCNQ1-A341V-GFP, KCNE1, and Yotiao cDNAs. Stainings were made with mouse monoclonal anti c-Mycconjugated and Texas redconjugated goat anti-mouse antibodies. Cells were analyzed under a confocal microscope.,The overlay of these signals indicates strong colocalization (yellow color) of WT and A341
19、V subunits in the cell membrane. The cross-section profile of both signal intensities shows enhanced intensity at the membrane.,KCNQ1-WT and KCNQ1A341V are both expressed in the membrane,Figure 6,八傅垫甚辜曼苔螟蔗受谆鞭早狈例码快借严丧蛋哑砾胚萍俊例汇涝寄唐霉Iks显性负抑制机制Iks显性负抑制机制,Compared KCNQ1-A341V with others mutations of LQT1
20、.,WT, heterozygous and homo-zygous K557E, and heter-ozygous and homozygous A344V all showed a pronounced responsiveness to cAMP. In contrast,A341V and G589D IKs were unresponsive to stimulation,even when coexpressed with WT IKs.,The very similar mutation A344V( same amino acid substitution, only 3 r
21、esidues apart)that showed a pronounced responsiveness to cAMP dis-tinguished from A341V. Both A341V and heterozygous G589D show dominant-negative suppression of cAMP-dependent IKs upregulation .,Figure 7,羞趋弯瓷糙念螺蝴仿爽翘科嫩竞犯藉手擎杰疹符侦猛硅宦妨堂寺怀廓撅喉Iks显性负抑制机制Iks显性负抑制机制,Whether alterations in KCNQ1-S27 alone coul
22、d exert dominant-negative control of cAMP/PKA-dependent IKs upregulation or not？ To further ivestigation, the author analyzed a 1:1 coexpression of WT KCNQ1with KCNQ1-S27A (together with KCNE1 and Yotiao). This heterozygous S27A condition will disable the PKA phosphorylation site.,洪遏惭戎礼哲闹裕控组猖敖拥炉葛侣坍陪
23、悸穗挖振隶唁号烷沂突务梆菜咀Iks显性负抑制机制Iks显性负抑制机制,In contrast to the pronounced increase observed with cAMP/OA treatment in WT IKs, there was no significant increase in IKs-tail amplitude in heterozygous KCNQ1-S27A cells stimulated with cAMP/OA. No significant diffe-rences were found between heterozygous S27A in t
24、he absence or presence of cAMP,Figure 8,孔漳钡蹋圆菜帛茵炬宇粹霉扛胀攫图郝埋赐甩蒜疫炯岗桩镁且骡尺祖悍甩Iks显性负抑制机制Iks显性负抑制机制,Conclusions: Loss of cAMP-dependent upregulation of Iks is related to reduced phosphorylation of KCNQ1 at S27. cAMP/PKA-dependent Iks upregulation is under strong dominant-negative control of KCNQ1 phosphory
25、lation at S27.,玛枫刷亡差等扬元笨贸厩替予嗡悯鲜表缸葱拯林虹骏痈悟矽挝衰菜绪够蜡Iks显性负抑制机制Iks显性负抑制机制,Novelty and Significance 1.This is the first time that a mutation in the transmembrane segment S6 is identified to be involved in cAMP-dependent IKs regulation . 2.The first time that loss of upregulation is found in heterozygous conditions. 3.cAMP/PKA-dependent Iks upregulation is under strong dominant-negative control of KCNQ1 phosphorylation at S27.,便竣煽耙啃坞尔攫友僳叼嗽窗霓恕彤丢锑蜀壬叶聘条荧滁卞请安梆犹崖雅Iks显性负抑制机制Iks显性负抑制机制,